Heterodimeric antibodies that bind prostate specific membrane antigen (psma) and cd3

ABSTRACT

Provided herein are novel antigen binding domains and antibodies (e.g., heterodimeric antibodies) that bind Prostate Specific Membrane Antigen (PSMA). In exemplary embodiments, the anti-PSMA antibodies also bind CD3. Such antibodies that bind PSMA and CD3 are useful, for example in the treatment of PSMA-related cancer.

PRIORITY

This application claims the benefit of U.S. Provisional PatentApplication Nos. 63/025,082, filed May 14, 2020 and 63/042,315, filedJun. 22, 2020 which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 21, 2021, isnamed 067461-5269-US_SL.txt and is 1,465,531 bytes in size.

BACKGROUND

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer. An increasingly prevalent avenuebeing explored is the engineering of single immunoglobulin moleculesthat co-engage two different antigens. Such alternate antibody formatsthat engage two different antigens are often referred to as bispecificantibodies. Because the considerable diversity of the antibody variableregion (Fv) makes it possible to produce an Fv that recognizes virtuallyany molecule, the typical approach to bispecific antibody generation isthe introduction of new variable regions into the antibody.

A particularly useful approach for bispecific antibodies is to engineera first binding domain which engages CD3 and a second binding domainwhich engages an antigen associated with or upregulated on cancer cellsso that the bispecific antibody redirects CD3⁺ T cells to destroy thecancer cells. Prostate cancer (PC) is one of the most prevalent cancersin men, and end stage (castration-resistant prostate cancer) has nocurative treatment option. Prostate Specific Membrane Antigen (PSMA), atype II transmembrane protein with a large extracellular domain, haslong generated interest as a therapeutic target. It is highlyoverexpressed in PC compared to normal tissue, and its expression hasbeen shown to correlate with malignancy. Previous attempts to targetPSMA include antibody-based radiotherapy and antibody drug conjugates,which have shown some success but can be hampered by the inherenttoxicity of the modality. Thus, there is a need for additional anti-PSMAantibodies for the treatment of PSMA-related cancers include, forexample, prostate cancer.

BRIEF SUMMARY

Provided herein are novel bispecific antibodies to CD3 and PSMA that arecapable of localizing CD3⁺ effector T cells to PSMA expressing tumorssuch as in prostate cancer. The anti-PSMA antibodies provided hereininclude PSMA binding domain with binding affinities and valencies thatallow for the advantageous selectivity for cells expressing high levelsof PSMA while minimizing reactivity on low PSMA expressing cells. Insome embodiments, such anti-PSMA antibodies include CD3 binding domainswith binding affinity that further contribute to selective targeting ofhigh-PSMA expressing cells lines. Such PSMA antibodies are useful, forexample, for cancers that express high levels of PSMA including, forexample, prostate cancer.

In one aspect, provided herein is a composition that includes a ProstateSpecific Membrane Antigen (PSMA) binding domain. The PSMA binding domainincludes: a) a variable heavy domain that includes the variable heavycomplementary determining regions 1-3 (vhCDR1-3) of PSMA-H variableheavy domain H1 (FIG. 17); and b) a variable light domain that includesthe variable light complementary determining regions (vlCDR1-3) of aPSMA-H variable light domain selected from PSMA-H variable light domainsL1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). In some embodiments, thevhCDR1-3 and vlCDR1-3 are selected from the vhCDR1-3 and vlCDR1-3sequences of any one of the anti-PSMA binding domains provided in FIGS.19A-19X.

Also provided herein are nucleic acid compositions that includepolynucleotide(s) encoding the subject PSMA binding domains, expressionvectors that include such polynucleotides and host cells that includesuch expression vectors. Also provided herein are methods of making suchPSMA binding domains.

In another aspect, provided herein is composition that includes aProstate Specific Membrane Antigen (PSMA) binding domain. The PSMAbinding domain includes: a) a variable heavy domain, wherein thevariable heavy domain is the PSMA-H variable heavy domain H1 (FIG. 17);and b) a variable light domain selected from PSMA-H variable lightdomains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E).

In another aspect, provided herein is an anti-PSMA antibody thatincludes a Prostate Specific Membrane Antigen (PSMA) binding domain. ThePSMA binding domain includes: a) a variable heavy domain that includesthe variable heavy complementary determining regions 1-3 (vhCDR1-3) ofPSMA-H variable heavy domain H1 (FIG. 17); and b) a variable lightdomain that includes the variable light complementary determiningregions (vlCDR1-3) of a PSMA-H variable light domain selected fromPSMA-H variable light domains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E).In some embodiments, the vhCDR1-3 and vlCDR1-3 are selected from thevhCDR1-3 and vlCDR1-3 of selected from any one of the anti-PSMA bindingdomains provided in FIGS. 19A-19X.

In one aspect, provided herein is a Prostate Specific Membrane Antigen(PSMA) binding domain. The PSMA binding domain includes: a) a variableheavy domain, wherein the variable heavy domain is the PSMA-H variableheavy domain H1 (FIG. 17); and b) a variable light domain selected fromPSMA-H variable light domains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E).In some embodiments, the antibody includes: a) a first monomer thatincludes a first antigen binding domain and a first constant domain; andb) a second monomer that includes a second antigen binding domain and asecond constant domain, wherein either of the first antigen bindingdomain or second antigen binding domain is the PSMA binding domain. Incertain embodiments, the first antigen binding domain and the secondantigen binding domain bind different antigens.

In some embodiments, the first antigen binding domain is the PSMAbinding domain and the second antigen binding domain is a CD3 bindingdomain. In particular embodiments, the CD3 binding domain includes thevhCDR1-3, and vlCDR1-3 of any of the following CD3 binding domains:H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47,H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89,L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F). In certainembodiments, the vhCDR1-3 and vlCDR1-3 of the CD3 binding domain areselected from the vhCDR1-3 and vlCDR1-3 in FIGS. 10A-10F. In someembodiments, the CD3 binding domain includes the variable heavy domainand variable light domain of any of the following CD3 binding domains:H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47,H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89,L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F). In certainembodiments, the CD3 binding domain is an anti-CD3 scFv. In someembodiments, the scFv includes a charged scFv linker. In someembodiments, the first and second constant domains each include CH2-CH3.In certain embodiments, the first and second constant domains eachinclude CH1-hinge-CH2-CH3.

In particular embodiments, the first and second constant domains eachare a variant constant domain. In some embodiments, the first and secondmonomers include a set of heterodimerization variants selected from thegroup consisting of those depicted in FIGS. 1A-1E. In exemplaryembodiments, the set of heterodimerization variants selected is from thegroup consisting of S364K/E357Q:L368D/K370S; S364K:L368D/K370S;S364K:L368E/K370S; D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V.In some embodiments, the first and second monomers each further includean ablation variant. In exemplary embodiments, the ablation variant isE233P/L234V/L235A/G236del/S267K.

In certain embodiments, at least one of the first or second monomerfurther includes one or more pI variants. In exemplary embodiments, theone or more pI variants is N208D/Q295E/N384D/Q418E/N421D.

In another aspect, provided is a heterodimeric antibody that includes:a) a first monomer, b) a second monomer; and c) a light chain. The firstmonomer includes: i) an anti-CD3 scFv that includes a first variableheavy domain, an scFv linker and a first variable light domain; and ii)a first Fc domain, wherein the scFv is covalently attached to theN-terminus of the first Fc domain using a domain linker. The secondmonomer includes a VH2-CH1-hinge-CH2-CH3 monomer, wherein VH is a secondvariable heavy domain and CH2-CH3 is a second Fc domain; and the lightchain includes a second variable light domain. The second variable heavydomain and the second variable light domain form an PSMA binding domain.

In some embodiments of the heterodimeric antibody, the second variableheavy domain includes the variable heavy complementary determiningregions 1-3 (vhCDR1-3) of PSMA-H variable heavy domain H1 (FIG. 17); andthe second variable light domain includes the variable lightcomplementary determining regions (vlCDR1-3) of a PSMA-H variable lightdomain selected from PSMA-H variable light domains L1 and L1.1-L1.84(FIGS. 17 and 18A-18E). In exemplary embodiments, the vhCDR1-3 of thesecond variable heavy domain and the vlCDR1-3 of the second variablelight domain are selected from any one of the anti-PSMA binding domainsprovided in FIGS. 19A-19X. In some embodiments, the second heavyvariable domain is PSMA-H variable heavy domain H1 (FIG. 17); and thesecond variable light domain is selected from PSMA-H variable lightdomains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E).

In certain embodiments, the anti-CD3 scFv includes the vhCDR1-3 and thevlCDR1-3 of any of the following CD3 binding domains: H1.30_L1.47,H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47,L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90,L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F). In some embodiments, thevhCDR1-3 and vlCDR1-3 of the anti-CD3 scFv are selected from thevhCDR1-3 and vlCDR1-3 in FIGS. 10A-10F. In exemplary embodiments, thefirst variable heavy domain and the first variable light domain are thevariable heavy domain and variable light domain, respectively, of any ofthe following CD3 binding domains: H1.30_L1.47, H1.32_L1.47,H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30,L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, andL1.47_H1.31 (FIGS. 10A-10F).

In one embodiment, the first variable light domain is covalentlyattached to the N-terminus of the first Fc domain using a domain linker.In some embodiments, the first variable heavy domain is covalentlyattached to the N-terminus of the first Fc domain using a domain linker.

In some embodiments of the heterodimeric antibody, the scFv linker is acharged scFv linker. In exemplary embodiments, the scFv linker is acharged scFv linker having the amino acid sequence (GKPGS)₄ (SEQ ID NO:1).

In certain embodiments, the first and second Fc domains are variant Fcdomains. In some embodiments, the first and second monomers include aset of heterodimerization variants selected from the group consisting ofthose depicted in FIGS. 1A-1E. In exemplary embodiments, the set ofheterodimerization variants selected is from the group consisting ofS364K/E357Q:L368D/K370S; S364K:L368D/K370S; S364K:L368E/K370S;D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V, wherein numberingis according to EU numbering. In some embodiments, the first and secondmonomers further includes an ablation variant. In exemplary embodiments,the ablation variant is E233P/L234V/L235A/G236del/S267K, whereinnumbering is according to EU numbering.

In some embodiments, one of the first or second monomer includes one ormore pI variants. In particular embodiments, the one or more pI variantsare N208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In exemplary embodiments of the heterodimeric antibody, the firstmonomer includes amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, the second monomer includesamino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the numbering is according to EU numbering.

In certain embodiments of the heterodimeric antibody, the first andsecond monomers each further include amino acid variants 428/434S.

In exemplary embodiments, the heterodimeric antibody is one of thefollowing heterodimeric antibodies: XENP14484, XENP33755, XENP33756,XENP33757, XENP33758, XENP33759, XENP33760, XENP33761, XENP33762,XENP34234, XENP34235, XENP34236, XENP16873, XENP16874, and XENP19722.

In another aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer; b) a second monomer; and c) a light chain.The first monomer includes, from N-terminus to C-terminus, ascFv-linker-CH2-CH3, wherein scFv is an anti-CD3 scFV and CH2-CH3 is afirst Fc domain. The second monomer includes, from N-terminus toC-terminus, a VH-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a second Fcdomain. The light chain includes a VL-CL. The first variant Fc domainincludes amino acid variants S364K/E357Q, the second variant Fc domainincludes amino acid variants L368D/K370S, the first and second variantFc domains each include amino acid variantsE233P/L234V/L235A/G236del/S267K, and the hinge-CH2-CH3 of the secondmonomer includes amino acid variants N208D/Q295E/N384D/Q418E/N421D. TheVH and VL form an PSMA binding domain that includes the variable heavydomain and the variable light domain, respectively, of an PSMA bindingdomain selected from PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11;PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68;PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81;PSMA-H H1_L1.84; and PSMA-H H1_L1.13. Further, the anti-CD3 scFvincludes the variable heavy domain and the variable light domain of aCD3 binding domain selected from H1.30_L1.47, H1.32_L1.47, H1.89_L1.47,H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30,L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31. Insuch heterodimeric antibodies, the numbering of the amino acid variantsis according to EU numbering.

In some embodiments of this heterodimeric antibody, the scFv includes acharged scFv linker having the amino acid sequence (GKPGS)₄ (SEQ ID NO:1). In certain embodiments, the first and second variant Fc domains eachfurther include amino acid variants 428/434S, wherein numbering isaccording to EU numbering.

In another aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer; b) a second monomer; and c) a common lightchain. The first monomer includes, from N-terminus to C-terminus, aVH1-CH1-linker 1-scFv-linker 2-CH2-CH3, wherein VH1 is a first variableheavy domain, scFv is an anti-CD3 scFV, linker 1 and linker 2 are afirst domain linker and second domain linker, respectively, and CH2-CH3is a first Fc domain. The second monomer includes, from N-terminus toC-terminus, a VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variableheavy domain and CH2-CH3 is a second Fc domain. The common light chainincludes a variable light domain. The first variable heavy domain andthe variable light domain form a first PSMA binding domain, and thesecond variable heavy domain and the variable light domain form a secondPSMA binding domain. In some embodiments, the first and second PSMAbinding domains each include the variable heavy complementarydetermining regions 1-3 (vhCDR1-3) of PSMA-H variable heavy domain H1(FIG. 17); and the variable light complementary determining regions(vlCDR1-3) of a PSMA-H variable light domain selected from PSMA-Hvariable light domains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). Inexemplary embodiments, the vhCDR1-3 and vlCDR1-3 of the first and secondPSMA binding domains are selected from the vhCDR1-3 and vlCDR1-3provided in FIGS. 17 and 18A-18E. In some embodiments, the first andsecond variable heavy domain each is a PSMA-H variable heavy domain H1(FIG. 17), and the variable light domain of the common light chain isselected from PSMA-H variable light domains L1 and L1.1-L1.84 (FIGS. 17and 18A-18E).

In certain embodiments, the scFv includes the vhCDR1-3 and the vlCDR1-3of any of the following CD3 binding domains: H1.30_L1.47, H1.32_L1.47,H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30,L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, andL1.47_H1.31 (FIGS. 10A-10F). In exemplary embodiments, the vhCDR1-3 andvlCDR1-3 of the scFv are selected from the vhCDR1-3 and vlCDR1-3 inFIGS. 10A-10F. In some embodiments, the scFv includes the variable heavydomain and variable light domain of any of the following CD3 bindingdomains: H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47,H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32,L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F).

In certain embodiments, the scFv includes an scFv variable heavy domain,an scFv variable light domain and an scFv linker that connects the scFvvariable heavy domain and the scFv variable light domain. In someembodiments, the scFv variable heavy domain is attached to theC-terminus of the CH1 of the first monomer using the first domain linkerand the scFv variable light domain is covalently attached to theN-terminus of the first Fc domain using the second domain linker. Inother embodiments, the scFv variable light domain is attached to theC-terminus of the CH1 of the first monomer using the first domain linkerand the scFv variable heavy domain is covalently attached to theN-terminus of the first Fc domain using the second domain linker. Incertain embodiments of this heterodimeric antibody, the scFv linker is acharged scFv linker. In exemplary embodiments, the scFv linker is acharged scFv linker having the amino acid sequence (GKPGS)₄ (SEQ ID NO:1).

In certain embodiments of this heterodimeric antibody, the first andsecond Fc domains are variant Fc domains. In some embodiments, the firstand second monomers include a set of heterodimerization variantsselected from the group consisting of those depicted in FIGS. 1A-1E. Inexemplary embodiments, the set of heterodimerization variants selectedis from the group consisting of S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In some embodiments, the first and second monomers further include anablation variant. In exemplary embodiments, the ablation variant isE233P/L234V/L235A/G236del/S267K, wherein numbering is according to EUnumbering.

In some embodiments, one of the first or second monomer includes one ormore pI variants. In particular embodiments, the one or more pI variantsare N208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In exemplary embodiments of the heterodimeric antibody, the firstmonomer includes amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, the second monomer includesamino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the numbering is according to EU numbering.

In certain embodiments of the heterodimeric antibody, the first andsecond monomers each further include amino acid variants 428/434S.

In exemplary embodiments, this heterodimeric antibody is one of thefollowing: XENP31602, XENP31603, XENP31855, XENP32218, XENP32219,XENP32220, XENP32221, XENP32222, XENP32223, XENP32224, XENP32225,XENP32226, XENP34237, XENP34238, XENP34239, XENP34625, XENP34626,XENP34627, XENP34628, XENP31853, XENP31856, XENP33063, XENP33064,XENP33065, XENP33066, XENP33067, XENP33068, XENP33069, XENP33070,XENP33071, XENP34240, XENP34241, XENP34242, XENP34629, XENP34630,XENP34631, XENP34632, XENP31854, and XENP31857.

In yet another aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer; b) a second monomer; and c) a common lightchain. The first monomer includes, from N-terminus to C-terminus, aVH-CH1-linker 1-scFv-linker 2-CH2-CH3, wherein scFv is an anti-CD3 scFVand CH2-CH3 is a first Fc domain. The b) a second monomer includes, fromN-terminus to C-terminus a VH-CH1-hinge-CH2-CH3, wherein CH2-CH3 is asecond Fc domain. The common light chain includes VL-CL. The firstvariant Fc domain includes amino acid variants S364K/E357Q, the secondvariant Fc domain includes amino acid variants L368D/K370S. The firstand second variant Fc domains each include amino acid variantsE233P/L234V/L235A/G236del/S267K, the hinge-CH2-CH3 of the second monomerincludes amino acid variants N208D/Q295E/N384D/Q418E/N421D. The VH ofthis heterodimeric antibody is PSMA-H variable heavy domain H1 (FIG.17), and the VL is a variable light domain selected from PSMA-H variablelight domains L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). Further, theanti-CD3 scFv includes the variable heavy domain and the variable lightdomain of a CD3 binding domain selected from H1.30_L1.47, H1.32_L1.47,H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30,L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, andL1.47_H1.31 (FIGS. 10A-10F). In such heterodimeric antibodies, thenumbering of the amino acid variants is according to EU numbering.

In some embodiments, the scFv includes a charged scFv linker having theamino acid sequence (GKPGS)₄ (SEQ ID NO: 1). In certain embodiments, thefirst and second variant Fc domains each further include amino acidvariants 428/434S.

In another aspect, provided herein are heterodimeric anti-PSMA×anti-CD3antibodies XENP14484, XENP33755, XENP33756, XENP33757, XENP33758,XENP33759, XENP33760, XENP33761, XENP33762, XENP34234, XENP34235,XENP34236, XENP16873, XENP16874, and XENP19722.

In yet another aspect, provided herein are heterodimericanti-PSMA×anti-CD3 antibodies XENP31602, XENP31603, XENP31855,XENP32218, XENP32219, XENP32220, XENP32221, XENP32222, XENP32223,XENP32224, XENP32225, XENP32226, XENP34237, XENP34238, XENP34239,XENP34625, XENP34626, XENP34627, XENP34628, XENP31853, XENP31856,XENP33063, XENP33064, XENP33065, XENP33066, XENP33067, XENP33068,XENP33069, XENP33070, XENP33071, XENP34240, XENP34241, XENP34242,XENP34629, XENP34630, XENP34631, XENP34632, XENP31854, and XENP31857.

Also provided herein are nucleic acid compositions that includepolynucleotide(s) encoding the subject anti-PSMA antibodies, expressionvectors that include such polynucleotides and host cells that includesuch expression vectors. Further provided herein are methods of makingsuch anti-PSMA antibodies, wherein a subject host cell is cultured underconditions wherein the anti-PSMA antibody is expressed, and recoveringthe anti-PSMA antibody.

In another aspect, provided herein is a method of treating a cancer thatincludes administering to a patient in need thereof any one of theanti-PSMA antibody described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1E depict useful pairs of Fc heterodimerization variant sets(including skew and pI variants). There are variants for which there areno corresponding “monomer 2” variants; these are pI variants which canbe used alone on either monomer.

FIG. 2 depicts a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theantibodies described herein (and other variant types as well, asoutlined herein).

FIG. 3 depicts useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer.

FIG. 4 depicts particularly useful embodiments of “non-Fv” components ofthe antibodies described herein.

FIG. 5 depicts a number of charged scFv linkers that find use inincreasing or decreasing the pI of the subject heterodimeric bsAbs thatutilize one or more scFv as a component, as described herein. The (+H)positive linker finds particular use herein, particularly with anti-CD3V_(L) and V_(H) sequences shown herein. A single prior art scFv linkerwith a single charge is referenced as “Whitlow”, from Whitlow et al.,Protein Engineering 6(8):989-995 (1993). It should be noted that thislinker was used for reducing aggregation and enhancing proteolyticstability in scFvs. Such charged scFv linkers can be used in any of thesubject antibody formats disclosed herein that include scFvs (e.g., 1+1Fab-scFv-Fc and 2+1 Fab₂-scFv-Fc formats).

FIG. 6 depicts a number of exemplary domain linkers. In someembodiments, these linkers find use linking a single-chain Fv to an Fcchain. In some embodiments, these linkers may be combined. For example,a GGGGS linker (SEQ ID NO: 2) may be combined with a “half hinge”linker.

FIGS. 7A-7D depict the sequences of several useful 1+1 Fab-scFv-Fcbispecific antibody format heavy chain backbones based on human IgG1,without the Fv sequences (e.g. the scFv and the VH for the Fab side).Backbone 1 is based on human IgG1 (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, C220S on the chain with theS364K/E357Q skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variantson the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 2 is based on human IgG1 (356E/358M allotype), and includesS364K:L368D/K370S skew variants, C220S on the chain with the S364K skewvariant, the N208D/Q295E/N384D/Q418E/N421D pI variants on the chain withL368D/K370S skew variants, and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Backbone 3 is based on human IgG1(356E/358M allotype), and includes S364K:L368E/K370S skew variants,C220S on the chain with the S364K skew variant, theN208D/Q295E/N384D/Q418E/N421D pI variants on the chain with L368E/K370Sskew variants and the E233P/L234V/L235A/G236del/S267K ablation variantson both chains. Backbone 4 is based on human IgG1 (356E/358M allotype),and includes D401K:K360E/Q362E/T411E skew variants, C220S on the chainwith the D401K skew variant, the N208D/Q295E/N384D/Q418E/N421D pIvariants on the chain with K360E/Q362E/T411E skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 5 is based on human IgG1 (356D/358L allotype), and includesS364K/E357Q:L368D/K370S skew variants, C220S on the chain with theS364K/E357Q skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variantson the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 6 is based on human IgG1 (356E/358M allotype), and includesS364K/E357Q:L368D/K370S skew variants, C220S on the chain with theS364K/E357Q skew variants, N208D/Q295E/N384D/Q418E/N421D pI variants onthe chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as an N297A variant on both chains. Backbone 7 is identical to 6except the mutation is N297S. Backbone 8 is based on human IgG4, andincludes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370Sskew variants, as well as a S228P (EU numbering, this is S241P in Kabat)variant on both chains that ablates Fab arm exchange as is known in theart. Backbone 9 is based on human IgG2, and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chain with L368D/K370S skew variants. Backbone 10 isbased on human IgG2, and includes the S364K/E357Q:L368D/K370S skewvariants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the chainwith L368D/K370S skew variants as well as a S267K variant on bothchains. Backbone 11 is identical to backbone 1, except it includesM428L/N434S Xtend mutations. Backbone 12 is based on human IgG1(356E/358M allotype), and includes S364K/E357Q:L368D/K370S skewvariants, C220S and the P217R/P229R/N276K pI variants on the chain withS364K/E357Q skew variants and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Included within each of thesebackbones are sequences that are 90, 95, 98 and 99% identical (asdefined herein) to the recited sequences, and/or contain from 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions (as comparedto the “parent” of the Figure, which, as will be appreciated by those inthe art, already contain a number of amino acid modifications ascompared to the parental human IgG1 (or IgG2 or IgG4, depending on thebackbone). That is, the recited backbones may contain additional aminoacid modifications (generally amino acid substitutions) in addition tothe skew, pI and ablation variants contained within the backbones ofthis figure.

FIGS. 8A-8C depict the sequences of several useful 2+1 Fab₂-scFv-Fcbispecific antibody format heavy chain backbones based on human IgG1,without the Fv sequences (e.g. the scFv and the VH for the Fab side).Backbone 1 is based on human IgG1 (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 2 is based on human IgG1 (356E/358M allotype), and includesS364K:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 3 is based on human IgG1 (356E/358M allotype), and includesS364K:L368E/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421D pIvariants on the chain with L368E/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 4 is based on human IgG1 (356E/358M allotype), and includesD401K:K360E/Q362E/T411E skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chain with K360E/Q362E/T411E skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 5 is based on human IgG1 (356D/358L allotype), and includesS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 6 is based on human IgG1 (356E/358M allotype), and includesS364K/E357Q:L368D/K370S skew variants, N208D/Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as an N297A variant on both chains. Backbone 7 is identical to 6except the mutation is N297S. Backbone 8 is identical to backbone 1,except it includes M428L/N434S Xtend mutations. Backbone 9 is based onhuman IgG1 (356E/358M allotype), and includes S364K/E357Q:L368D/K370Sskew variants, the P217R/P229R/N276K pI variants on the chain withS364K/E357Q skew variants and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Included within each of thesebackbones are sequences that are 90, 95, 98 and 99% identical (asdefined herein) to the recited sequences, and/or contain from 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions (as comparedto the “parent” of the Figure, which, as will be appreciated by those inthe art, already contain a number of amino acid modifications ascompared to the parental human IgG1 (or IgG2 or IgG4, depending on thebackbone). That is, the recited backbones may contain additional aminoacid modifications (generally amino acid substitutions) in addition tothe skew, pI and ablation variants contained within the backbones ofthis figure.

FIG. 9 depicts the sequences of several useful constant light domainbackbones based on human IgG1, without the Fv sequences (e.g. the scFvor the Fab). Included herein are constant light backbone sequences thatare 90, 95, 98 and 99% identical (as defined herein) to the recitedsequences, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10additional amino acid modifications.

FIGS. 10A-10F depict sequences for exemplary anti-CD3 scFvs suitable foruse in the bispecific antibodies described herein. The CDRs areunderlined, the scFv linker is double underlined (in the sequences, thescFv linker is a positively charged scFv (GKPGS)₄ linker (SEQ ID NO: 1),although as will be appreciated by those in the art, this linker can bereplaced by other linkers, including uncharged or negatively chargedlinkers, some of which are depicted in FIG. 5), and the slashes indicatethe border(s) of the variable domains. In addition, the namingconvention illustrates the orientation of the scFv from N- toC-terminus. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table2, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the V_(H) and V_(L) domains using othernumbering systems. Furthermore, as for all the sequences in the Figures,these V_(H) and V_(L) sequences can be used either in a scFv format orin a Fab format.

FIGS. 11A and 11B depict the antigen sequences for PSMA, includinghuman, mouse and cyno, to facilitate the development of antigen bindingdomains that bind to both for ease of clinical development.

FIG. 12 depicts illustrative IHC of biopsy cores of prostate cancer andadjacent normal tissue showing PSMA expression.

FIG. 13 depicts breakdown of IHC scores of 192 biopsy cores showing PSMAexpression.

FIG. 14 depicts antigen density (determined using QuickCal protocol) oncancer cell lines LnCAP, 22Rv1, Huh-7, A549, ASPC-1, HT29, and SKOV3 aswell as PSMA-transfected PC3 cells.

FIG. 15 depicts illustrative IHC of cancer cell lines andPSMA-transfected PC3 cells showing PSMA expression.

FIG. 16 depicts PSMA expression on cancer cell lines andPSMA-transfected PC3 cell lines as determined by IHC and flow cytometry.

FIG. 17 depicts the variable heavy and variable light chain sequencesfor an exemplary humanized PSMA binding domain referred to herein asPSMA-H, as well as the sequences for XENP31858 and XENP31604, anti-PSMAmAbs based on PSMA-H and IgG1 backbone. CDRs are underlined and slashesindicate the border(s) between the variable regions and constant domain.As noted herein and is true for every sequence herein containing CDRs,the exact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 2, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the V_(H) and V_(L) domains using other numberingsystems. Furthermore, as for all the sequences in the Figures, theseV_(H) and V_(L) sequences can be used either in a scFv format or in aFab format.

FIGS. 18A-18E depict the variable light chain sequences for PSMA-Hvariants engineered with the aim to tune binding affinity for humanPSMA. CDRs are underlined and slashes indicate the border(s) between thevariable regions and constant domain. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 2, and thus included herein are not only the CDRsthat are underlined but also CDRs included within V_(L) domains usingother numbering systems. Further, as for all the sequences in theFigures, these V_(L) sequences can be used either in a scFv format or ina Fab format. Each of the variable light domains depicted herein can bepaired with any other αPSMA variable heavy domain.

FIGS. 19A-19Y depict the amino acid sequences for PSMA-H variantsengineered with the aim to tune binding affinity for human PSMAformatted as bivalent anti-PSMA mAbs. CDRs are underlined and slashesindicate the border(s) between the variable regions and constant domain.As noted herein and is true for every sequence herein containing CDRs,the exact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 2, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the V_(H) and V_(L) domains using other numberingsystems.

FIG. 20A-20B depicts BLI-response, apparent dissociation constant(K_(Dapp)), association rate (k_(a)), and dissociation rate (k_(d)) ofaffinity-engineered PSMA-H variants (in bivalent IgG1 format) asdetermined by Octet. Substitutions in variable light regions are basedon Kabat numbering.

FIG. 21A-21B depicts a couple of formats of the present invention. FIG.21A depicts the “1+1 Fab-scFv-Fc” format, with a first Fab arm bindingPSMA and a second scFv arm binding CD3. FIG. 21B depicts the “2+1Fab₂-scFv-Fc” format, with a first Fab arm binding PSMA and a secondFab-scFv arm, wherein the Fab binds PSMA and the scFv binds CD3.

FIGS. 22A-22F depict the sequences for illustrative αPSMA×αCD3 bsAbs inthe 1+1 Fab-scFv-Fc format and comprising a H1.30_L1.47 anti-CD3 scFv(a.k.a. CD3 High[VHVL]). CDRs are underlined and slashes indicate theborder(s) between the variable regions and other chain components (e.g.constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 23 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the1+1 Fab-scFv-Fc format and comprising a H1.33_L1.47 anti-CD3 scFv(a.k.a. CD3 Intermediate[VHVL]). CDRs are underlined and slashesindicate the border(s) between the variable regions and other chaincomponents (e.g. constant region and domain linkers). It should be notedthat the αPSMA×αCD3 bsAbs can utilize variable region, Fc region, andconstant domain sequences that are 90, 95, 98 and 99% identical (asdefined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions. In addition, each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 24 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the1+1 Fab-scFv-Fc format and comprising a H1.31_L1.47 anti-CD3 scFv(a.k.a. CD3 High-Int[VHVL]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 25 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the1+1 Fab-scFv-Fc format and comprising a H1.32_L1.47 anti-CD3 scFv(a.k.a. CD3 High-Int #1[VHVL]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 26 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the2+1 Fab₂-scFv-Fc format and comprising a H1.30_L1.47 anti-CD3 scFv(a.k.a. CD3 High [VHVL]). CDRs are underlined and slashes indicate theborder(s) between the variable regions and other chain components (e.g.constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 27 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the2+1 Fab₂-scFv-Fc format and comprising a H1.32_L1.47 anti-CD3 scFv(a.k.a. CD3 High-Int #1[VHVL]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIGS. 28A-28K depict the sequences for illustrative αPSMA×αCD3 bsAbs inthe 2+1 Fab_(z)-scFv-Fc format and comprising a L1.47_H1.32 anti-CD3scFv (a.k.a. CD3 High-Int #1[VLVH]). CDRs are underlined and slashesindicate the border(s) between the variable regions and other chaincomponents (e.g. constant region and domain linkers). It should be notedthat the αPSMA×αCD3 bsAbs can utilize variable region, Fc region, andconstant domain sequences that are 90, 95, 98 and 99% identical (asdefined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions. In addition, each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 29 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the2+1 Fab₂-scFv-Fc format and comprising a H1.89_L1.47 anti-CD3 scFv(a.k.a. CD3 High-Int #2[VHVL]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIGS. 30A-30K depicts the sequences for illustrative αPSMA×αCD3 bsAbs inthe 2+1 Fab₂-scFv-Fc format and comprising a L1.47_H1.89 anti-CD3 scFv(a.k.a. CD3 High-Int #2[VLVH]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 31 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the2+1 Fab-scFv-Fc format and comprising a H1.33_L1.47 anti-CD3 scFv(a.k.a. CD3 Intermediate[VHVL]). CDRs are underlined and slashesindicate the border(s) between the variable regions and other chaincomponents (e.g. constant region and domain linkers). It should be notedthat the αPSMA×αCD3 bsAbs can utilize variable region, Fc region, andconstant domain sequences that are 90, 95, 98 and 99% identical (asdefined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions. In addition, each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 32 depicts the sequences for illustrative αPSMA×αCD3 bsAbs in the2+1 Fab-scFv-Fc format and comprising a L1.47_H1.33 anti-CD3 scFv(a.k.a. CD3 Intermediate[VLVH]). CDRs are underlined and slashesindicate the border(s) between the variable regions and other chaincomponents (e.g. constant region and domain linkers). It should be notedthat the αPSMA×αCD3 bsAbs can utilize variable region, Fc region, andconstant domain sequences that are 90, 95, 98 and 99% identical (asdefined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions. In addition, each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 33 depicts the sequences for illustrative prototypic αPSMA×αCD3bsAbs in alternative formats.

FIG. 34 depicts BLI-response, dissociation constant (K_(D)), associationrate (k_(a)), and dissociation rate (k_(d)) of affinity-engineeredPSMA-H variants (in the context of PSMA×CD3 bispecifics in the 1+1Fab-scFv-Fc format with a H1.30_L1.47 anti-CD3 scFv) for human PSMA asdetermined by Octet. Substitutions in variable light regions are basedon Kabat numbering.

FIG. 35 depicts BLI-response, dissociation constant (K_(D)), associationrate (k_(a)), and dissociation rate (k_(d)) of affinity-engineeredPSMA-H variants (in the context of PSMA×CD3 bispecifics in the 1+1Fab-scFv-Fc format with a H1.30_L1.47 anti-CD3 scFv) for cynomolgus PSMAas determined by Octet. Substitutions in variable light regions arebased on Kabat numbering. A couple of the bsAbs are listed with NA dueto odd sensorgrams.

FIG. 36 depicts induction of RTCC on luciferase-transduced PC3 cellswith varying surface PSMA densities by A) XENP34282 and B) XENP14484.The data show that the two prototypic 1+1 anti-PSMA×anti-CD3 inducedRTCC of cell lines expressing high and low PSMA levels, including PC3(˜3K) which represent normal tissues, with similar potency.

FIG. 37 depicts induction of T cell proliferation (as indicated bypercentage T cells expressing Ki67) by A) XENP34282 and B) XENP14484 inthe presence of PC3 cells with varying surface PSMA densities. The datashow that the two prototypic 1+1 anti-PSMA×anti-CD3 induced T cellproliferation in the presence of cell lines expressing high and low PSMAlevels, including PC3 (˜3K) which represent normal tissues, with similarpotency.

FIG. 38 depicts the binding to PSMA-transfected PC3 (˜32K) cells(representative of low PSMA-expressing on-target cells) by A) 1+1Fab-scFv-Fc bispecific antibodies XENP14484, XENP33756, XENP33757,XENP33761, and XENP337652 and by B) 2+1 Fab2-scFv-Fc bispecificantibodies XENP31620, XENP32218, XENP32220, XENP32224, and XENP32225.The data show that as monovalent PSMA binding affinity is decreased inthe 1+1 Fab-scFv-Fc bispecific antibodies, their binding to PC3 (˜32K)cells is drastically reduced. Notably, as monovalent PSMA bindingaffinity is decreased in the 2+1 Fab2-scFv-Fc bispecific antibodies,their binding to PC3 (˜32K) cells is retained.

FIG. 39 depicts induction of RTCC on transduced PC3luciferase-transfected cancer cells with varying surface PSMA densitiesby A) XENP32218, B) XENP32220, and C) XENP32224.

FIG. 40 depicts induction of T cell proliferation (as indicated bypercentage T cells expressing Ki67) by A) XENP32218, B) XENP32220, andC) XENP32224 in the presence of cancer cells with varying surface PSMAdensities.

FIG. 41 depicts induction of T cell degranulation (as indicated bypercentage T cells expressing CD107a) by A) XENP32218, B) XENP32220, andC) XENP32224 in the presence of cancer cells with varying surface PSMAdensities.

FIG. 42 depicts induction of RTCC on luciferase-transduced PC3 cancercells with varying surface PSMA densities by XENP31855 (1 nM K_(D)PSMA+CD3 High-Int#1(VLVH) in 2+1 Fab-scFv-Fc format).

FIG. 43 depicts induction of T cell degranulation (as indicated bypercentage T cells expressing CD107a) by XENP31856 (1 nM K_(D) PSMA+CD3High-Int#2(VLVH)) in the presence of PC3 cancer cells transfected withvarying surface PSMA densities. The data show that XENP31856 was highlyselective for high PSMA expressing PC3 (˜100K) cell line; however,XENP31856 induced little to no degranulation in the presence of all thelow PSMA expressing PC3 cell lines.

FIG. 44 depicts induction of RTCC on luciferase-transduced PC3 cancercells with varying surface PSMA densities by XENP33063 (7 nM K_(D)PSMA+CD3 High-Int#2(VLVH)). The data show that XENP33063 was highlyselective for high PSMA expressing PC3 (˜100K) cell line; however,XENP31856 induced little to no killing on all the low PSMA expressingPC3 cell lines.

FIG. 45 depicts induction of RTCC on luciferase-transduced LNCaP cancercells and 22Rv1 cancer cells by XENP32220 (38 nM K_(D) PSMA+CD3High-Int#1(VLVH)). The data show that XENP32220 was able to induce cellkill on both LNCaP and 22Rv1 cancer cells.

FIG. 46 depicts induction of T cell degranulation (as indicated bypercentage T cells expressing CD107a) by A) XENP14484, B) XENP34282, andC) XENP34283 in the presence of cancer cells with varying surface PSMAdensities. The data show that the three prototypic 1+1anti-PSMA×anti-CD3 induced T cell degranulation in the presence of celllines expressing higher and lower PSMA levels with similar potency.

FIG. 47 depicts tumor volume on Day 19 in PSMA-transfected PC3 (˜100K)and huPBMC-engrafted mice following first dose with PBS, bivalentanti-PD1 mAb, XENP32218, XENP32220, or XENP32224. Each of the tunedPSMA×CD3 bispecific antibodies significantly enhanced (p<0.05 vs. PBS orαPD-1 mAb) anti-tumor activity (as indicated by tumor volume; statisticsperformed on baseline corrected data using unpaired t-test).

FIG. 48 depicts the change in tumor volume (as determined by calipermeasurements) over time in PSMA-transfected PC3 (˜100K) andhuPBMC-engrafted mice dosed with PBS, bivalent anti-PD1 mAb, XENP32218,XENP32220, or XENP32224.

FIGS. 49A-49C depict the pharmacokinetic data from a study in which eachhealthy male cynomolgus was administered by IV either a 1× dose, 10×dose, or 60× dose of the indicated test article. Xtend variantsXENP34262, XENP34267, and XENP34628 showed improved pharmacokineticsover non-Xtend variants XENP32218, XENP32220, and XENP32224. All testarticles were tolerated at each dose level.

FIGS. 50A-50K depict several formats for use in the anti-PSMA×anti-CD3bispecific antibodies disclosed herein. The first is the “1+1Fab-scFv-Fc” format (also referred to as the “bottle opener” or “TripleF” format), with a first antigen binding domain that is a Fab domain anda second anti-antigen binding domain that is an scFv domain (FIG. 1A).Additionally, “mAb-Fv,” “mAb-scFv,” “2+1 Fab2-scFv-Fc” (also referred toas the “central scFv” or “central-scFv” format”), “central-Fv,” “onearmed central-scFv,” “one scFv-mAb,” “scFv-mAb,” “dual scFv,” “trident,”and non-heterodimeric bispecific formats are all shown. The scFv domainsdepicted in FIGS. 10A-10F can be either, from N- to C-terminusorientation: variable heavy-(optional linker)-variable light, orvariable light-(optional linker)-variable heavy. In addition, for theone armed scFv-mAb, the scFv can be attached either to the N-terminus ofa heavy chain monomer or to the N-terminus of the light chain. Incertain embodiments, “Anti-antigen 1” in FIG. 50 refers to a PSMAbinding domain. In certain embodiments, “Anti-antigen 1” in FIG. 50refers to a CD3 binding domain. In certain embodiments, “Anti-antigen 2”in FIG. 50 refers to a PSMA binding domain. In certain embodiments“Anti-antigen 2” in FIG. 50 refers to a CD3 binding domain. In someembodiments, “Anti-antigen 1” in FIG. 50 refers to a PSMA binding domainand “Anti-antigen 2” in FIG. 50 refers to a CD3 binding domain. In someembodiments, “Anti-antigen 1” in FIG. 50 refers to a CD3 binding domainand “Anti-antigen 2” in FIG. 50 refers to a PSMA binding domain. Any ofthe PSMA binding domains and CD3 binding domains disclosed can beincluded in the bispecific formats of FIG. 50.

DETAILED DESCRIPTION I. Overview

Provided herein are novel anti-CD3×anti-PSMA (also referred to asanti-PSMA×anti-CD3, αCD3×αPSMA, or αPSMA×αCD3) heterodimeric bispecificantibodies and methods of using such antibodies for the treatment ofcancers. In particular, provided herein are anti-CD3, anti-PSMAbispecific antibodies in a variety of formats such as those depicted inFIGS. 21A and 21B. These bispecific antibodies are useful for thetreatment of cancers, particularly those with increased PSMA expressionsuch as prostate cancers. Such antibodies are used to direct CD3+effector T cells to PSMA+ tumors, thereby allowing the CD3+ effector Tcells to attack and lyse the PSMA+ tumors.

The anti-PSMA antibodies provided herein include PSMA binding domainwith binding affinities and valencies that allow for the advantageousselectivity for cells expressing high levels of PSMA while minimizingreactivity on low PSMA expressing cells. Such PSMA antibodies areuseful, for example, for cancers that express high levels of PSMAincluding, for example, prostate cancer.

In some embodiments, such anti-PSMA antibodies include CD3 bindingdomains with binding affinity that further contribute to selectivetargeting of high-PSMA expressing cells lines. Such bispecificantibodies that have different binding affinities to human CD3 that canalter or reduce the potential side effects of anti-CD3 therapy. That is,in some embodiments the antibodies described herein provide antibodyconstructs comprising anti-CD3 antigen binding domains that are “strong”or “high affinity” binders to CD3 (e.g. one example are heavy and lightvariable domains depicted as H1.30_L1.47 (optionally including a chargedlinker as appropriate)) and also bind to PSMA. In other embodiments, theantibodies described herein provide antibody constructs comprisinganti-CD3 antigen binding domains that are “lite” or “lower affinity”binders to CD3. Additional embodiments provides antibody constructscomprising anti-CD3 antigen binding domains that have intermediate or“medium” affinity to CD3 that also bind to PSMA. While a very largenumber of anti-CD3 antigen binding domains (ABDs) can be used,particularly useful embodiments use 6 different anti-CD3 ABDs, althoughthey can be used in two scFv orientations as discussed herein. Affinityis generally measured using a Biacore assay.

It should be appreciated that the “high, medium, low” anti-CD3 sequencesprovided herein can be used in a variety of heterodimerization formatsas depicted in FIGS. 21A, 21B. In general, due to the potential sideeffects of T cell recruitment, exemplary embodiments utilize formatsthat only bind CD3 monovalently, such as depicted in FIGS. 21A and 21B,and in the formats depicted herein, it is the CD3 ABD that is a scFv asmore fully described herein. In contrast, the subject bispecificantibodies can bind PSMA either monovalently (e.g. FIG. 21A) orbivalently (e.g. FIG. 21B).

Accordingly, in one aspect, provided herein are heterodimeric antibodiesthat bind to two different antigens, e.g. the antibodies are“bispecific”, in that they bind two different target antigens, generallyPSMA and CD3 as described herein. These heterodimeric antibodies canbind these target antigens either monovalently (e.g. there is a singleantigen binding domain such as a variable heavy and variable lightdomain pair) or bivalently (there are two antigen binding domains thateach independently bind the antigen). In some embodiments, theheterodimeric antibody provided herein includes one CD3 binding domainand one PSMA binding domain (e.g., heterodimeric antibodies in the “1+1Fab-scFv-Fc” format described herein). In other embodiments, theheterodimeric antibody provided herein includes one CD3 binding domainand two PSMA binding domains (e.g., heterodimeric antibodies in the “2+1Fab₂-scFv-Fc” formats described herein). The heterodimeric antibodiesprovided herein are based on the use different monomers which containamino acid substitutions that “skew” formation of heterodimers overhomodimers, as is more fully outlined below, coupled with “pI variants”that allow simple purification of the heterodimers away from thehomodimers, as is similarly outlined below. The heterodimeric bispecificantibodies provided generally rely on the use of engineered or variantFc domains that can self-assemble in production cells to produceheterodimeric proteins, and methods to generate and purify suchheterodimeric proteins.

II. Nomenclature

The antibodies provided herein are listed in several different formats.In some instances, each monomer of a particular antibody is given aunique “XENP” number, although as will be appreciated in the art, alonger sequence might contain a shorter one. For example, a “scFv-Fc”monomer of a 1+1 Fab-scFv-Fc format antibody may have a first XENPnumber, while the scFv domain itself will have a different XENP number.Some molecules have three polypeptides, so the XENP number, with thecomponents, is used as a name. Thus, the molecule XENP31602, which is in2+1 Fab₂-scFv-Fc format, comprises three sequences (see FIG. 26): 1) a“Fab-Fc Heavy Chain” monomer; 2) a “Fab-scFv-Fc Heavy Chain” monomer;and 3) a “Light Chain” monomer or equivalents, although one of skill inthe art would be able to identify these easily through sequencealignment. These XENP numbers are in the sequence listing as well asidentifiers, and used in the Figures. In addition, one molecule,comprising the three components, gives rise to multiple sequenceidentifiers. For example, the listing of the Fab includes, the fullheavy chain sequence, the variable heavy domain sequence and the threeCDRs of the variable heavy domain sequence, the full light chainsequence, a variable light domain sequence and the three CDRs of thevariable light domain sequence. A Fab-scFv-Fc monomer includes a fulllength sequence, a variable heavy domain sequence, 3 heavy CDRsequences, and an scFv sequence (include scFv variable heavy domainsequence, scFv variable light domain sequence and scFv linker). Notethat some molecules herein with a scFv domain use a single charged scFvlinker (+H), although others can be used. In addition, the namingnomenclature of particular antigen binding domains (e.g., PSMA and CD3binding domains) use a “Hx.xx_Ly.yy” type of format, with the numbersbeing unique identifiers to particular variable chain sequences. Thus,the PSMA binding domain PSMA-H H1_L1 (e.g., FIG. 17A) is “H1_L1”, whichindicates that the variable heavy domain, H1, was combined with thelight domain L1. In the case that these sequences are used as scFvs, thedesignation “H1_L1”, indicates that the variable heavy domain, H1 iscombined with the light domain, L1, and is in VH-linker-VL orientation,from N- to C-terminus. This molecule with the identical sequences of theheavy and light variable domains but in the reverse order (VL-linker-VHorientation, from N- to C-terminus) would be designated “L1_H1.1”.Similarly, different constructs may “mix and match” the heavy and lightchains as will be evident from the sequence listing and the figures.

III. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “PSMA” or “Prostate Specific Membrane Antigen” (e.g., GenebankAccession Number NP 005012.2) herein is meant a type II transmembraneprotein that is expressed in all prostatic tissues, including primaryprostate adenocarcinomas, metastatic prostate cancer, and in the tumorneovasculature of many solid tumors. In prostate cancer (PCa), PSMA ishighly expressed in poorly differentiated, highly metastatic prostaticcells and in castrate-resistant models. PSMA sequences are disclosed inFIGS. 11A and 11B.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with more than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore, SPR or BLI assay. Ofparticular use in the ablation of FcγR binding are those shown in FIG.5, which generally are added to both monomers.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecificphagocytic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause phagocytosis of the target cell.

As used herein, term “antibody” is used generally. Antibodies describedherein can take on a number of formats as described herein, includingtraditional antibodies as well as antibody derivatives, fragments andmimetics, described herein.

Traditional immunoglobulin (Ig) antibodies are “Y” shaped tetramers.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light chain” monomer(typically having a molecular weight of about 25 kDa) and one “heavychain” monomer (typically having a molecular weight of about 50-70 kDa).

Other useful antibody formats include, but are not limited to, the 1+1Fab-scFv-Fc format and 2+1 Fab₂-scFv-Fc antibody formats describedherein, as well as “mAb-Fv,” “mAb-scFv,” “central-Fv”, “one armedscFv-mAb,” “scFv-mAb,” “dual scFv,” and “trident” format antibodies, asshown in FIG. 50.

Antibody heavy chains typically include a variable heavy (VH) domain,which includes vhCDR1-3, and an Fc domain, which includes a CH2-CH3monomer. In some embodiments, antibody heavy chains include a hinge andCH1 domain. Traditional antibody heavy chains are monomers that areorganized, from N- to C-terminus: VH-CH1-hinge-CH2-CH3. TheCH1-hinge-CH2-CH3 is collectively referred to as the heavy chain“constant domain” or “constant region” of the antibody, of which thereare five different categories or “isotypes”: IgA, IgD, IgG, IgE and IgM.Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses. Forexample, as shown in US Publication 2009/0163699, incorporated byreference, the antibodies described herein include the use of humanIgG1/G2 hybrids.

In some embodiments, the antibodies provided herein include IgG isotypeconstant domains, which has several subclasses, including, but notlimited to IgG1, IgG2, IgG3, and IgG4. In the IgG subclass ofimmunoglobulins, there are several immunoglobulin domains in the heavychain. By “immunoglobulin (Ig) domain” herein is meant a region of animmunoglobulin having a distinct tertiary structure. Of interest in theantibodies described herein are the heavy chain domains, including, theconstant heavy (CH) domains and the hinge domains. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-220 according to the EU index as in Kabat. “CH2” refers topositions 237-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat. Asshown herein and described below, the pI variants can be in one or moreof the CH regions, as well as the hinge region, discussed below.

It should be noted that IgG1 has different allotypes with polymorphismsat 356 (D or E) and 358 (L or M). The sequences depicted herein use the356D/358M allotype, however the other allotype is included herein. Thatis, any sequence inclusive of an IgG1 Fc domain included herein can have356E/358L replacing the 356D/358M allotype. It should be understood thattherapeutic antibodies can also comprise hybrids of isotypes and/orsubclasses. For example, as shown in US Publication 2009/0163699,incorporated by reference, the present antibodies, in some embodiments,include IgG1/IgG2 hybrids.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody, in someinstances, excluding all of the first constant region immunoglobulindomain (e.g., CH1) or a portion thereof, and in some cases, optionallyincluding all or part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all ora portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, insome cases, the Fc domain includes, from N- to C-terminal, CH2-CH3 andhinge-CH2-CH3. In some embodiments, the Fc domain is that from IgG1,IgG2, IgG3 or IgG4, with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3finding particular use in many embodiments. Additionally, in the case ofhuman IgG1 Fc domains, frequently the hinge includes a C220S amino acidsubstitution. Furthermore, in the case of human IgG4 Fc domains,frequently the hinge includes a S228P amino acid substitution. Althoughthe boundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues E216, C226, or A231 to itscarboxyl-terminal, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR or to the FcRn.

By “heavy chain constant region” herein is meant the CH1-hinge-CH2-CH3portion of an antibody (or fragments thereof), excluding the variableheavy domain; in EU numbering of human IgG1 this is amino acids 118-447By “heavy chain constant region fragment” herein is meant a heavy chainconstant region that contains fewer amino acids from either or both ofthe N- and C-termini but still retains the ability to form a dimer withanother heavy chain constant region.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “hinge domain”herein is meant the flexible polypeptide comprising the amino acidsbetween the first and second constant domains of an antibody.Structurally, the IgG CH1 domain ends at EU position 215, and the IgGCH2 domain begins at residue EU position 231. Thus for IgG the antibodyhinge is herein defined to include positions 216 (E216 in IgG1) to 230(p230 in IgG1), wherein the numbering is according to the EU index as inKabat. In some cases, a “hinge fragment” is used, which contains feweramino acids at either or both of the N- and C-termini of the hingedomain. As noted herein, pI variants can be made in the hinge region aswell. Many of the antibodies herein have at least one the cysteines atposition 220 according to EU numbering (hinge region) replaced by aserine. Generally, this modification is on the “scFv monomer” side formost of the sequences depicted herein, although it can also be on the“Fab monomer” side, or both, to reduce disulfide formation. Specificallyincluded within the sequences herein are one or both of these cysteinesreplaced (C220S).

As will be appreciated by those in the art, the exact numbering andplacement of the heavy constant region domains can be different amongdifferent numbering systems. A useful comparison of heavy constantregion numbering according to EU and Kabat is as below, see Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, entirelyincorporated by reference.

TABLE 1 EU Numbering Kabat Numbering CH1 118-215 114-223 Hinge 216-230226-243 CH2 231-340 244-360 CH3 341-447 361-478

The antibody light chain generally comprises two domains: the variablelight domain (VL), which includes light chain CDRs vlCDR1-3, and aconstant light chain region (often referred to as CL or Cκ. The antibodylight chain is typically organized from N- to C-terminus: VL-CL.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen (e.g., PSMA orCD3) as discussed herein. As is known in the art, these CDRs aregenerally present as a first set of variable heavy CDRs (vhCDRs orVHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), eachcomprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy CDRs andvlCDR1, vlCDR2 and vlCDR3 vhCDR3 variable light CDRs. The CDRs arepresent in the variable heavy domain (vhCDR1-3) and variable lightdomain (vlCDR1-3). The variable heavy domain and variable light domainfrom an Fv region.

The antibodies described herein provide a large number of different CDRsets. In this case, a “full CDR set” comprises the three variable lightand three variable heavy CDRs, e.g., a vlCDR1, vlCDR2, vlCDR3, vhCDR1,vhCDR2 and vhCDR3. These can be part of a larger variable light orvariable heavy domain, respectfully. In addition, as more fully outlinedherein, the variable heavy and variable light domains can be on separatepolypeptide chains, when a heavy and light chain is used (for examplewhen Fabs are used), or on a single polypeptide chain in the case ofscFv sequences.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1,vhCDR2 and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3). A usefulcomparison of CDR numbering is as below, see Lafranc et al., Dev. Comp.Immunol. 27(1):55-77 (2003):

TABLE 2 Kabat + Chothia IMGT Kabat AbM Chothia Contact Xencor vhCDR126-35   27-38  31-35  26-35  26-32  30-35   27-35  vhCDR2 50-65   56-65 50-65  50-58  52-56  47-58   54-61  vhCDR3 95-102 105-117 95-102 95-10295-102 93-101 103-116 vlCDR1 24-34   27-38  24-34  24-34  24-34  30-36  27-38  vlCDR2 50-56   56-65  50-56  50-56  50-56  46-55   56-62  vlCDR389-97  105-117 89-97  89-97  89-97  89-96   97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of the antigen binding domains andantibodies. “Epitope” refers to a determinant that interacts with aspecific antigen binding site in the variable region of an antibodymolecule known as a paratope. Epitopes are groupings of molecules suchas amino acids or sugar side chains and usually have specific structuralcharacteristics, as well as specific charge characteristics. A singleantigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the disclosure not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

In some embodiments, the six CDRs of the antigen binding domain arecontributed by a variable heavy and a variable light domain. In a “Fab”format, the set of 6 CDRs are contributed by two different polypeptidesequences, the variable heavy domain (vh or VH; containing the vhCDR1,vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containingthe vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domainbeing attached to the N-terminus of the CH1 domain of the heavy chainand the C-terminus of the vl domain being attached to the N-terminus ofthe constant light domain (and thus forming the light chain). In a scFvformat, the vh and vl domains are covalently attached, generally throughthe use of a linker (a “scFv linker”) as outlined herein, into a singlepolypeptide sequence, which can be either (starting from the N-terminus)vh-linker-vl or vl-linker-vh, with the former being generally preferred(including optional domain linkers on each side, depending on the formatused (e.g., from FIG. 1). In general, the C-terminus of the scFv domainis attached to the N-terminus of the hinge in the second monomer.

By “variable region” or “variable domain” as used herein is meant theregion of an immunoglobulin that comprises one or more Ig domainssubstantially encoded by any of the Vκ, Vλ, and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively, and contains the CDRs that confer antigen specificity.Thus, a “variable heavy domain” pairs with a “variable light domain” toform an antigen binding domain (“ABD”). In addition, each variabledomain comprises three hypervariable regions (“complementary determiningregions,” “CDRs”) (VHCDR1, VHCDR2 and VHCDR3 for the variable heavydomain and VLCDR1, VLCDR2 and VLCDR3 for the variable light domain) andfour framework (FR) regions, arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described in Table 2.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains, generally ontwo different polypeptide chains (e.g. VH-CH1 on one chain and VL-CL onthe other). Fab may refer to this region in isolation, or this region inthe context of a bispecific antibody described herein. In the context ofa Fab, the Fab comprises an Fv region in addition to the CH1 and CLdomains.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of an ABD. Fv regionscan be formatted as both Fabs (as discussed above, generally twodifferent polypeptides that also include the constant regions asoutlined above) and scFvs, where the VL and VH domains are combined(generally with a linker as discussed herein) to form an scFv.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (VH-linker-VL orVL-linker-VH). In the sequences depicted in the sequence listing and inthe figures, the order of the VH and VL domain is indicated in the name,e.g. H.X_L.Y means N- to C-terminal is VH-linker-VL, and L.Y_H.X isVL-linker-VH.

Some embodiments of the subject antibodies provided herein comprise atleast one scFv domain, which, while not naturally occurring, generallyincludes a variable heavy domain and a variable light domain, linkedtogether by a scFv linker. As outlined herein, while the scFv domain isgenerally from N- to C-terminus oriented as VH-scFv linker-VL, this canbe reversed for any of the scFv domains (or those constructed using vhand vl sequences from Fabs), to VL-scFv linker-VH, with optional linkersat one or both ends depending on the format.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. The protein variant has atleast one amino acid modification compared to the parent protein, yetnot so many that the variant protein will not align with the parentalprotein using an alignment program such as that described below. Ingeneral, variant proteins (such as variant Fc domains, etc., outlinedherein, are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% identical to the parent protein, using the alignmentprograms described below, such as BLAST. “Variant” as used herein alsorefers to particular amino acid modifications that confer particularfunction (e.g., a “heterodimerization variant,” “pI variant,” “ablationvariant,” etc.).

As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4,although human sequences with variants can also serve as “parentpolypeptides”, for example the IgG1/2 hybrid of US Publication2006/0134105 can be included. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain ascompared to an Fc domain of human IgG1, IgG2 or IgG4.

“Fc variant” or “variant Fc” as used herein is meant a proteincomprising an amino acid modification in an Fc domain. The modificationcan be an addition, deletion, or substitution. The Fc variants aredefined according to the amino acid modifications that compose them.Thus, for example, N434S or 434S is an Fc variant with the substitutionfor serine at position 434 relative to the parent Fc polypeptide,wherein the numbering is according to the EU index. Likewise,M428L/N434S defines an Fc variant with the substitutions M428L and N434Srelative to the parent Fc polypeptide. The identity of the WT amino acidmay be unspecified, in which case the aforementioned variant is referredto as 428L/434S. It is noted that the order in which substitutions areprovided is arbitrary, that is to say that, for example, 428L/434S isthe same Fc variant as 434S/428L, and so on. For all positions discussedherein that relate to antibodies or derivatives and fragments thereof(e.g., Fc domains), unless otherwise noted, amino acid positionnumbering is according to the EU index. The “EU index” or “EU index asin Kabat” or “EU numbering” scheme refers to the numbering of the EUantibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, herebyentirely incorporated by reference).

In general, variant Fc domains have at least about 80, 85, 90, 95, 97,98 or 99 percent identity to the corresponding parental human IgG Fcdomain (using the identity algorithms discussed below, with oneembodiment utilizing the BLAST algorithm as is known in the art, usingdefault parameters). Alternatively, the variant Fc domains can have from1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 amino acid modifications as compared to theparental Fc domain. Alternatively, the variant Fc domains can have up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 amino acid modifications as compared to theparental Fc domain. Additionally, as discussed herein, the variant Fcdomains described herein still retain the ability to form a dimer withanother Fc domain as measured using known techniques as describedherein, such as non-denaturing gel electrophoresis.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. In addition, polypeptides that make up the antibodiesdescribed herein may include synthetic derivatization of one or moreside chains or termini, glycosylation, PEGylation, circular permutation,cyclization, linkers to other molecules, fusion to proteins or proteindomains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the human IgGs comprise a serine at position 434, the substitution434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life. An “FcRn variant” is one thatincreases binding to the FcRn receptor, and suitable FcRn variants areshown below.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Accordingly, by“parent immunoglobulin” as used herein is meant an unmodifiedimmunoglobulin polypeptide that is modified to generate a variant, andby “parent antibody” as used herein is meant an unmodified antibody thatis modified to generate a variant antibody. It should be noted that“parent antibody” includes known commercial, recombinantly producedantibodies as outlined below. In this context, a “parent Fc domain” willbe relative to the recited variant; thus, a “variant human IgG1 Fcdomain” is compared to the parent Fc domain of human IgG1, a “varianthuman IgG4 Fc domain” is compared to the parent Fc domain human IgG4,etc.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the antigen binding domain comprising the variableregions of a given antibody.

By “strandedness” in the context of the monomers of the heterodimericantibodies described herein is meant that, similar to the two strands ofDNA that “match”, heterodimerization variants are incorporated into eachmonomer so as to preserve the ability to “match” to form heterodimers.For example, if some pI variants are engineered into monomer A (e.g.making the pI higher) then steric variants that are “charge pairs” thatcan be utilized as well do not interfere with the pI variants, e.g. thecharge variants that make a pI higher are put on the same “strand” or“monomer” to preserve both functionalities. Similarly, for “skew”variants that come in pairs of a set as more fully outlined below, theskilled artisan will consider pI in deciding into which strand ormonomer one set of the pair will go, such that pI separation ismaximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “host cell” in the context of producing a bispecific antibodyaccording to the antibodies described herein is meant a cell thatcontains the exogeneous nucleic acids encoding the components of thebispecific antibody and is capable of expressing the bispecific antibodyunder suitable conditions. Suitable host cells are discussed below.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

Provided herein are a number of antibody domains that have sequenceidentity to human antibody domains. Sequence identity between twosimilar sequences (e.g., antibody variable domains) can be measured byalgorithms such as that of Smith, T. F. & Waterman, M. S. (1981)“Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homologyalgorithm]; Needleman, S. B. & Wunsch, C D. (1970) “A General MethodApplicable To The Search For Similarities In The Amino Acid Sequence OfTwo Proteins,” J. Mol. Biol. 48:443 [homology alignment algorithm],Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For BiologicalSequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [searchfor similarity method]; or Altschul, S. F. et al, (1990) “Basic LocalAlignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm,see https://blast.ncbi.nlm.nih.gov/Blast.cgi. When using any of theaforementioned algorithms, the default parameters (for Window length,gap penalty, etc) are used. In one embodiment, sequence identity is doneusing the BLAST algorithm, using default parameters

The antibodies described herein are generally isolated or recombinant.“Isolated,” when used to describe the various polypeptides disclosedherein, means a polypeptide that has been identified and separatedand/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody that is substantially free of other antibodies having differentantigenic specificities. “Recombinant” means the antibodies aregenerated using recombinant nucleic acid techniques in exogeneous hostcells, and they can be isolated as well.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where K_(D) refers to a dissociationrate of a particular antibody-antigen interaction. Typically, anantibody that specifically binds an antigen will have a KD that is 20-,50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for acontrol molecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacore, SPRor BLI assay.

IV. PSMA Binding Domains

In one aspect, provided herein are PSMA antigen binding domains (ABDs)and compositions that include such PSMA antigen binding domains (ABDs),including anti-PSMA antibodies. Subject antibodies that include suchPSMA antigen binding domains (e.g., anti-PSMA×anti-CD3 bispecificantibodies) advantageously target cells that express high levels of PSMAover those that express levels of PSMA (e.g., normal cells). Such PSMAbinding domains and related antibodies find use, for example, in thetreatment of PSMA associated cancers, such as prostate cancer.

As will be appreciated by those in the art, suitable PSMA bindingdomains can comprise a set of 6 CDRs as depicted in the sequence listingand FIGS. 17-19, either as the CDRs are underlined or, in the case wherea different numbering scheme is used as described herein and as shown inTable 2, as the CDRs that are identified using other alignments withinthe variable heavy (VH) domain and variable light domain (VL) sequencesof those depicted in FIGS. 17-19 and the Sequence Listing (see Table 2).Suitable PSMA ABDs can also include the entire VH and VL sequences asdepicted in these sequences and figures, used as scFvs or as Fabdomains.

In one embodiment, the PSMA antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of a PSMA ABD described herein, includingthe Figures and sequence listing. In some embodiments, the PSMA ABDincludes the vhCDR1-3 of PSMA-H H1 (FIG. 17) and the vlCDR1-3 of a PSMAvariable light domain selected from PSMA-H L1 (FIG. 17) and L1.1-L1.84(FIG. 18A-E). In exemplary embodiments, the PSMA ABD is one of thefollowing PSMA ABDs: PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11;PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68;PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81;PSMA-H H1_L1.84; and PSMA-H H1_L1.13.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to PSMA, provided herein are variantPSMA ABDS having CDRs that include at least one modification of the PSMAABD CDRs disclosed herein. In one embodiment, the PSMA ABD includes aset of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidmodifications as compared to the 6 CDRs of a PSMA ABD described herein,including the figures and sequence listing. In exemplary embodiments,the PSMA ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10amino acid modifications as compared to the 6 CDRs of one of thefollowing PSMA ABDs: PSMA ABDs: PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-HH1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-HH1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-HH1_L1.81; PSMA-H H1_L1.84; and PSMA-H H1_L1.13. In certain embodiments,the variant PSMA ABD is capable of binding PSMA antigen, as measured byat least one of a Biacore, surface plasmon resonance (SPR) and/or BLI(biolayer interferometry, e.g., Octet assay) assay, with the latterfinding particular use in many embodiments. In particular embodiments,the PSMA ABD is capable of binding human PSMA antigen (see Example 5).

In one embodiment, the PSMA ABD includes 6 CDRs that are at least 90,95, 97, 98 or 99% identical to the 6 CDRs of a PSMA ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the PSMA ABD includes 6 CDRs that are at least 90, 95, 97,98 or 99% identical to the 6 CDRs of one of the following PSMA ABDs:PSMA ABDs: PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; and PSMA-H H1_L1.13. In certain embodiments, the PSMA ABD iscapable of binding to PSMA antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the PSMAABD is capable of binding human PSMA antigen (see FIG. 2).

In another exemplary embodiment, the PSMA ABD include the variable heavy(VH) domain and variable light (VL) domain of any one of the PSMA ABDsdescribed herein, including the figures and sequence listing. In someembodiments, the PSMA ABD includes the PSMA-H H1 variable heavy domain(FIG. 17) and a variable light domain selected from PSMA-H L1 (FIG. 17)and L1.1-L1.84 (FIG. 18A-E). In exemplary embodiments, the PSMA ABD isone of the following PSMA ABDs: PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84(FIGS. 17-19). In exemplary embodiments, the PSMA ABD is PSMA-H H1_L1,PSMA-H H1_L1.58. PSMA-H H1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26;PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52;PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-H H1_L1.84; or PSMA-H H1_L1.13(FIGS. 17-19).

In addition to the parental PSMA variable heavy and variable lightdomains disclosed herein, provided herein are PSMA ABDs that include avariable heavy domain and/or a variable light domain that are variantsof a PSMA ABD VH and VL domain disclosed herein. In one embodiment, thevariant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid changes from a VH and/or VL domain of a PSMA ABD describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of oneof the following PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84 (FIGS. 17-19). Inexemplary embodiments, the PSMA ABD is PSMA-H H1_L1, PSMA-H H1_L1.58;PSMA-H H1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75;PSMA-H H1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78;PSMA-H H1_L1.81; PSMA-H H1_L1.84; or PSMA-H H1_L1.13 (FIGS. 17-19). Incertain embodiments, the PSMA ABD is capable of binding to PSMA, asmeasured at least one of a Biacore, surface plasmon resonance (SPR)and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with thelatter finding particular use in many embodiments. In particularembodiments, the PSMA ABD is capable of binding human PSMA antigen (seeExample 5).

In one embodiment, the variant VH and/or VL domain is at least 90, 95,97, 98 or 99% identical to the VH and/or VL of a PSMA ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98or 99% identical to the VH and/or VL of one of the following PSMA ABDs:PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84 (FIGS. 17-19). In exemplaryembodiments, the PSMA ABD is PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-HH1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-HH1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-HH1_L1.81; PSMA-H H1_L1.84; or PSMA-H H1_L1.13 (FIGS. 17-19). In certainembodiments, the PSMA ABD is capable of binding to the PSMA, as measuredby at least one of a Biacore, surface plasmon resonance (SPR) and/or BLI(biolayer interferometry, e.g., Octet assay) assay, with the latterfinding particular use in many embodiments. In particular embodiments,the PSMA ABD is capable of binding human PSMA antigen (see Example 5).

V. Antibodies

In one aspect, provided herein are antibodies that bind to PSMA (e.g.,anti-PSMA antibodies). In certain embodiments, the antibody binds tohuman PSMA (FIG. 11A). Subject anti-PSMA antibodies include monospecificPSMA antibodies, as well as multi-specific (e.g., bispecific) anti-PSMAantibodies. In certain embodiments, the anti-PSMA antibody has a formataccording to any one of the antibody formats depicted in FIGS. 21A and21B.

In some embodiments, the subject compositions include a PSMA bindingdomain. In some embodiments, the composition includes an antibody havinga PSMA binding domain. Antibodies provided herein include one, two,three, four, and five or more PSMA binding domains. In certainembodiments, the PSMA binding domain includes any one of the vhCDR1,vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of an PSMA bindingdomain selected from those depicted in FIGS. 17-19. In some embodiments,the PSMA binding domain includes the underlined vhCDR1, vhCDR2, vhCDR3,vlCDR1, vlCDR2 and vlCDR3 sequences of a PSMA binding domain selectedfrom those depicted in FIGS. 17-19. In some embodiments, the PSMAbinding domain includes the variable heavy domain and variable lightdomain of a PSMA binding domain selected from those depicted in FIGS.17-19. PSMA binding domains depicted in FIGS. 12, 13A-13B, and 14A-14Iinclude: PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84.

In one aspect, provided herein are bispecific antibodies that bind toPSMA and CD3, in various formats as outlined below, and generallydepicted in FIGS. 21A and 21B. These bispecific, heterodimericantibodies include a PSMA binding domain. In certain embodiments, thePSMA binding domain includes the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2and VLCDR3 sequences of an PSMA binding domain selected from the groupconsisting of those depicted in FIGS. 17-19. In some embodiments, thePSMA binding domain includes the underlined VHCDR1, VHCDR2, VHCDR3,VLCDR1, VLCDR2 and VLCDR3 sequences of an PSMA binding domain selectedfrom those depicted in FIGS. 17-19.

These bispecific heterodimeric antibodies bind PSMA and CD3. Suchantibodies include a CD3 binding domain and at least one PSMA bindingdomain. Any suitable PSMA binding domain can be included in theanti-PSMA×anti-CD3 bispecific antibody. In some embodiments, theanti-PSMA×anti-CD3 bispecific antibody includes one, two, three, four ormore PSMA binding domains, including but not limited to those depictedin FIGS. 17-19. In certain embodiments, the anti-PSMA×anti-CD3 antibodyincludes an PSMA binding domain that includes the VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2 and VLCDR3 sequences of an PSMA binding domainselected from the group consisting of PSMA-H H1_L1 and PSMA-HH1_L1.1-L1.84 (FIGS. 17-19). In some embodiments, the anti-PSMA×anti-CD3antibody includes a PSMA binding domain that includes the underlinedVHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 sequences of an PSMAbinding domain selected from the group consisting of PSMA-H H1_L1 andPSMA-H H1_L1.1-L1.84 (FIGS. 17-19). In some embodiments, theanti-PSMA×anti-CD3 antibody includes a PSMA binding domain that includesthe variable heavy domain and variable light domain of an PSMA bindingdomain selected from the group consisting of PSMA-H H1_L1 and PSMA-HH1_L1.1-L1.84 (FIGS. 17-19).

The anti-PSMA×anti-CD3 antibody provided herein can include any suitableCD3 binding domain. In certain embodiments, the anti-PSMA×anti-CD3antibody includes a CD3 binding domain that includes the VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2 and VLCDR3 sequences of a CD3 binding domainselected from the group consisting of those depicted in FIG. 10A-F. Insome embodiments, the anti-PSMA×anti-CD3 antibody includes a CD3 bindingdomain that includes the underlined VHCDR1, VHCDR2, VHCDR3, VLCDR1,VLCDR2 and VLCDR3 sequences of a CD3 binding domain selected from thegroup consisting of those depicted in FIGS. 10A-10F. In someembodiments, the anti-PSMA×anti-CD3 antibody includes a CD3 bindingdomain that includes the variable heavy domain and variable light domainof a CD3 binding domain selected from the group consisting of thosedepicted in FIGS. 10A-10F. In some embodiments, the CD3 binding domainis selected from H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47,H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32,L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F).As outlined herein, these anti-CD3 antigen binding domains (CD3-ABDs)can be used in scFv formats in either orientation (e.g. from N- toC-terminal, VH-scFv linker-VL or VL-scFv linker-VH).

The antibodies provided herein include different antibody domains. Asdescribed herein and known in the art, the antibodies described hereininclude different domains within the heavy and light chains, which canbe overlapping as well. These domains include, but are not limited to,the Fc domain, the CH1 domain, the CH2 domain, the CH3 domain, the hingedomain, the heavy constant domain (CH1-hinge-Fc domain orCH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, Fab domains and scFv domains.

As shown herein, there are a number of suitable linkers (for use aseither domain linkers or scFv linkers) that can be used to covalentlyattach the recited domains (e.g., scFvs, Fabs, Fc domains, etc.),including traditional peptide bonds, generated by recombinanttechniques. Exemplary linkers to attach domains of the subject antibodyto each other are depicted in FIG. 6. In some embodiments, the linkerpeptide may predominantly include the following amino acid residues:Gly, Ser, Ala, or Thr. The linker peptide should have a length that isadequate to link two molecules in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity. In one embodiment, the linker is from about 1 to 50amino acids in length, preferably about 1 to 30 amino acids in length.In one embodiment, linkers of 1 to 20 amino acids in length may be used,with from about 5 to about 10 amino acids finding use in someembodiments. Useful linkers include glycine-serine polymers, includingfor example (GS)n, (GSGGS)n (SEQ ID NO: 3), (GGGGS)n (SEQ ID NO: 627),and (GGGS)n (SEQ ID NO: 4), where n is an integer of at least one (andgenerally from 3 to 4), glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers, some of which are shown in FIG. 5and FIG. 6. Alternatively, a variety of nonproteinaceous polymers,including but not limited to polyethylene glycol (PEG), polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol, may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. For example, in FIG. 21B, theremay be a domain linker that attaches the C-terminus of the CH1 domain ofthe Fab to the N-terminus of the scFv, with another optional domainlinker attaching the C-terminus of the scFv to the CH2 domain (althoughin many embodiments the hinge is used as this domain linker). While anysuitable linker can be used, many embodiments utilize a glycine-serinepolymer as the domain linker, including for example (GS)n, (GSGGS)n (SEQID NO: 3), (GGGGS)n (SEQ ID NO: 627), and (GGGS)n (SEQ ID NO: 4), wheren is an integer of at least one (and generally from 3 to 4 to 5) as wellas any peptide sequence that allows for recombinant attachment of thetwo domains with sufficient length and flexibility to allow each domainto retain its biological function. In some cases, and with attentionbeing paid to “strandedness”, as outlined below, charged domain linkers,as used in some embodiments of scFv linkers can be used. Exemplaryuseful domain linkers are depicted in FIG. 6.

With particular reference to the domain linker used to attach the scFvdomain to the Fc domain in the “2+1” format, there are several domainlinkers that find particular use, including “full hinge C220S variant,”“flex half hinge,” “charged half hinge 1,” and “charged half hinge 2” asshown in FIG. 6.

In some embodiments, the linker is a “scFv linker”, used to covalentlyattach the VH and VL domains as discussed herein. In many cases, thescFv linker is a charged scFv linker, a number of which are shown inFIG. 5. Accordingly, in some embodiments, the antibodies describedherein further provide charged scFv linkers, to facilitate theseparation in pI between a first and a second monomer. That is, byincorporating a charged scFv linker, either positive or negative (orboth, in the case of scaffolds that use scFvs on different monomers),this allows the monomer comprising the charged linker to alter the pIwithout making further changes in the Fc domains. These charged linkerscan be substituted into any scFv containing standard linkers. Again, aswill be appreciated by those in the art, charged scFv linkers are usedon the correct “strand” or monomer, according to the desired changes inpI. For example, as discussed herein, to make 1+1 Fab-scFv-Fc formatheterodimeric antibody, the original pI of the Fv region for each of thedesired antigen binding domains are calculated, and one is chosen tomake an scFv, and depending on the pI, either positive or negativelinkers are chosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the antibodies described herein as well, and thus thoseincluded in FIG. 5 can be used in any embodiment herein where a linkeris utilized.

In particular, the formats depicted in FIGS. 21A and 21B are antibodies,usually referred to as “heterodimeric antibodies”, meaning that theprotein has at least two associated Fc sequences self-assembled into aheterodimeric Fc domain and at least two Fv regions, whether as Fabs oras scFvs.

The PSMA binding domains provided can be included in any useful antibodyformat including, for example, canonical immunoglobulin, as well as the1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fv formats provided herein. Otheruseful antibody formats include, but are not limited to, “mAb-Fv,”“mAb-scFv,” “central-Fv”, “one armed scFv-mAb,” “scFv-mAb,” “dual scFv,”and “trident” format antibodies, as disclosed in FIGS. 50A-50K.

In some embodiments, the subject antibody includes one or more of thePSMA ABDs provided herein. In some embodiments, the antibody includesone PSMA ABD. In other embodiments, the antibody includes two PSMA ABDs.In exemplary embodiments, the PSMA ABD includes the variable heavydomain and variable light domain of one of the following PSMA ABDs:PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84 (FIGS. 17-19). In someembodiments, the PSMA ABD is one of the following PSMA ABDs: PSMA-HH1_L1 and PSMA-H H1_L1.1-L1.84 (FIGS. 17-19).

In an exemplary embodiment, the antibody is a bispecific antibody thatincludes one or two PSMA ABDs, including any of the PSMA ABDs providedherein. Bispecific antibody that include such PSMA ABDs include, forexample, 1+1 Fab-scFv-Fc and 2+1 Fab₂-scFv-Fc bispecifics formatantibodies. In exemplary embodiments, the PSMA ABD is one of thefollowing PSMA-H H1_L1 and PSMA-H H1_L1.1-L1.84 (FIGS. 17-19). Inexemplary embodiments the PSMA binding domains is a Fab. In someembodiments, such bispecific antibodies are heterodimeric bispecificantibodies that include any of the heterodimerization skew variants, pIvariants and/or ablation variants described herein.

A. Chimeric and Humanized Antibodies

In certain embodiments, the antibodies described herein comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody (using the methods outlined herein). A humanantibody that is “the product of” or “derived from” a particular humangermline immunoglobulin sequence may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a humanized antibody typically is atleast 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the antibody as being derived from humansequences when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a humanized antibody may be at least 95, 96, 97, 98 or 99%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence tothe amino acid sequence encoded by the germline immunoglobulin gene.Typically, a humanized antibody derived from a particular human germlinesequence will display no more than 10-20 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene(prior to the introduction of any skew, pI and ablation variants herein;that is, the number of variants is generally low, prior to theintroduction of the variants described herein). In certain cases, thehumanized antibody may display no more than 5, or even no more than 4,3, 2, or 1 amino acid difference from the amino acid sequence encoded bythe germline immunoglobulin gene (again, prior to the introduction ofany skew, pI and ablation variants herein; that is, the number ofvariants is generally low, prior to the introduction of the variantsdescribed herein).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

B. Heterodimeric Antibodies

In exemplary embodiments, the bispecific antibodies provided herein areheterodimeric bispecific antibodies that include two variant Fc domainsequences. Such variant Fc domains include amino acid modifications tofacilitate the self-assembly and/or purification of the heterodimericantibodies.

An ongoing problem in antibody technologies is the desire for“bispecific” antibodies that bind to two different antigenssimultaneously, in general thus allowing the different antigens to bebrought into proximity and resulting in new functionalities and newtherapies. In general, these antibodies are made by including genes foreach heavy and light chain into the host cells. This generally resultsin the formation of the desired heterodimer (A-B), as well as the twohomodimers (A-A and B-B (not including the light chain heterodimericissues)). However, a major obstacle in the formation of bispecificantibodies is the difficulty in biasing the formation of the desiredheterodimeric antibody over the formation of the homodimers and/orpurifying the heterodimeric antibody away from the homodimers.

There are a number of mechanisms that can be used to generate thesubject heterodimeric antibodies. In addition, as will be appreciated bythose in the art, these different mechanisms can be combined to ensurehigh heterodimerization. Amino acid modifications that facilitate theproduction and purification of heterodimers are collectively referred togenerally as “heterodimerization variants.” As discussed below,heterodimerization variants include “skew” variants (e.g., the “knobsand holes” and the “charge pairs” variants described below) as well as“pI variants,” which allow purification of heterodimers from homodimers.As is generally described in U.S. Pat. No. 9,605,084, herebyincorporated by reference in its entirety and specifically as below forthe discussion of heterodimerization variants, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”) as described inU.S. Pat. No. 9,605,084, “electrostatic steering” or “charge pairs” asdescribed in U.S. Pat. No. 9,605,084, pI variants as described in U.S.Pat. No. 9,605,084, and general additional Fc variants as outlined inU.S. Pat. No. 9,605,084 and below.

Heterodimerization variants that are useful for the formation andpurification of the subject heterodimeric antibody (e.g., bispecificantibodies) are further discussed in detailed below.

1. Skew Variants

In some embodiments, the heterodimeric antibody includes skew variantswhich are one or more amino acid modifications in a first Fc domain (A)and/or a second Fc domain (B) that favor the formation of Fcheterodimers (Fc dimers that include the first and the second Fc domain;(A-B) over Fc homodimers (Fc dimers that include two of the first Fcdomain or two of the second Fc domain; A-A or B-B). Suitable skewvariants are included in the FIG. 29 of US Publ. App. No. 2016/0355608,hereby incorporated by reference in its entirety and specifically forits disclosure of skew variants, as well as in FIGS. 1A-1E and FIG. 4.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

In some embodiments, the skew variants advantageously and simultaneouslyfavor heterodimerization based on both the “knobs and holes” mechanismas well as the “electrostatic steering” mechanism. In some embodiments,the heterodimeric antibody includes one or more sets of suchheterodimerization skew variants. These variants come in “pairs” of“sets”. That is, one set of the pair is incorporated into the firstmonomer and the other set of the pair is incorporated into the secondmonomer. It should be noted that these sets do not necessarily behave as“knobs in holes” variants, with a one-to-one correspondence between aresidue on one monomer and a residue on the other. That is, these pairsof sets may instead form an interface between the two monomers thatencourages heterodimer formation and discourages homodimer formation,allowing the percentage of heterodimers that spontaneously form underbiological conditions to be over 90%, rather than the expected 50% (25%homodimer A/A:50% heterodimer A/B:25% homodimer B/B). Exemplaryheterodimerization “skew” variants are depicted in FIG. 4. In exemplaryembodiments, the heterodimeric antibody includes aS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L; K370S:S364K/E357Q; ora T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) “skew” variant amino acidsubstitution set. In an exemplary embodiment, the heterodimeric antibodyincludes a “S364K/E357Q:L368D/K370S” amino acid substitution set. Interms of nomenclature, the pair “S364K/E357Q:L368D/K370S” means that oneof the monomers includes an Fc domain that includes the amino acidsubstitutions S364K and E357Q and the other monomer includes an Fcdomain that includes the amino acid substitutions L368D and K370S; asabove, the “strandedness” of these pairs depends on the starting pI.

In some embodiments, the skew variants provided herein can be optionallyand independently incorporated with any other modifications, including,but not limited to, other skew variants (see, e.g., in FIG. 37 of USPubl. App. No. 2012/0149876, herein incorporated by reference,particularly for its disclosure of skew variants), pI variants, isotypicvariants, FcRn variants, ablation variants, etc. into one or both of thefirst and second Fc domains of the heterodimeric antibody. Further,individual modifications can also independently and optionally beincluded or excluded from the subject the heterodimeric antibody.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the antibodies described herein.

A list of suitable skew variants is found in FIGS. 1A-1E, with FIG. 4showing some pairs of particular utility in many embodiments. Ofparticular use in many embodiments are the pairs of sets including, butnot limited to, S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S364K/E357Q. In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers has the doublevariant set S364K/E357Q and the other has the double variant setL368D/K370S.

2. pI (Isoelectric Point) Variants for Heterodimers

In some embodiments, the heterodimeric antibody includes purificationvariants that advantageously allow for the separation of heterodimericantibody (e.g., anti-PSMA×anti-CD3 bispecific antibody) from homodimericproteins.

There are several basic mechanisms that can lead to ease of purifyingheterodimeric antibodies. For example, modifications to one or both ofthe antibody heavy chain monomers A and B such that each monomer has adifferent pI allows for the isoelectric purification of heterodimericA-B antibody from monomeric A-A and B-B proteins. Alternatively, somescaffold formats, such as the “1+1 Fab-scFv-Fc” format and the “2+1Fab₂-scFv-Fc” format, also allows separation on the basis of size. Asdescribed above, it is also possible to “skew” the formation ofheterodimers over homodimers using skew variants. Thus, a combination ofheterodimerization skew variants and pI variants find particular use inthe heterodimeric antibodies provided herein.

Additionally, as more fully outlined below, depending on the format ofthe heterodimeric antibody, pI variants either contained within theconstant region and/or Fc domains of a monomer, and/or domain linkerscan be used. In some embodiments, the heterodimeric antibody includesadditional modifications for alternative functionalities that can alsocreate pI changes, such as Fc, FcRn and KO variants.

In some embodiments, the subject heterodimeric antibodies providedherein include at least one monomer with one or more modifications thatalter the pI of the monomer (i.e., a “pI variant”). In general, as willbe appreciated by those in the art, there are two general categories ofpI variants: those that increase the pI of the protein (basic changes)and those that decrease the pI of the protein (acidic changes). Asdescribed herein, all combinations of these variants can be done: onemonomer may be wild type, or a variant that does not display asignificantly different pI from wild-type, and the other can be eithermore basic or more acidic. Alternatively, each monomer is changed, oneto more basic and one to more acidic.

Depending on the format of the heterodimer antibody, pI variants can beeither contained within the constant and/or Fc domains of a monomer, orcharged linkers, either domain linkers or scFv linkers, can be used.That is, antibody formats that utilize scFv(s) such as “1+1Fab-scFv-Fc”, format can include charged scFv linkers (either positiveor negative), that give a further pI boost for purification purposes. Aswill be appreciated by those in the art, some 1+1 Fab-scFv-Fc formatsare useful with just charged scFv linkers and no additional pIadjustments, although the antibodies described herein do provide pIvariants that are on one or both of the monomers, and/or charged domainlinkers as well. In addition, additional amino acid engineering foralternative functionalities may also confer pI changes, such as Fc, FcRnand KO variants.

In subject heterodimeric antibodies that utilizes pI as a separationmechanism to allow the purification of heterodimeric proteins, aminoacid variants are introduced into one or both of the monomerpolypeptides. That is, the pI of one of the monomers (referred to hereinfor simplicity as “monomer A”) can be engineered away from monomer B, orboth monomer A and B change be changed, with the pI of monomer Aincreasing and the pI of monomer B decreasing. As is outlined more fullybelow, the pI changes of either or both monomers can be done by removingor adding a charged residue (e.g., a neutral amino acid is replaced by apositively or negatively charged amino acid residue, e.g., glycine toglutamic acid), changing a charged residue from positive or negative tothe opposite charge (aspartic acid to lysine) or changing a chargedresidue to a neutral residue (e.g., loss of a charge; lysine to serine).A number of these variants are shown in the FIGS. 3 and 4.

Thus, in some embodiments, the subject heterodimeric antibody includesamino acid modifications in the constant regions that alter theisoelectric point (pI) of at least one, if not both, of the monomers ofa dimeric protein to form “pI antibodies”) by incorporating amino acidsubstitutions (“pI variants” or “pI substitutions”) into one or both ofthe monomers. As shown herein, the separation of the heterodimers fromthe two homodimers can be accomplished if the pIs of the two monomersdiffer by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 orgreater all finding use in the antibodies described herein.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components, for example in the1+1 Fab-scFv-Fc and 2+1 Fab₂-scFv-Fc formats, the starting pI of thescFv and Fab(s) of interest. That is, to determine which monomer toengineer or in which “direction” (e.g., more positive or more negative),the Fv sequences of the two target antigens are calculated and adecision is made from there. As is known in the art, different Fvs willhave different starting pIs which are exploited in the antibodiesdescribed herein. In general, as outlined herein, the pIs are engineeredto result in a total pI difference of each monomer of at least about 0.1logs, with 0.2 to 0.5 being preferred as outlined herein.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying bispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and pI heterodimerization variants) are notincluded in the variable regions, such that each individual antibodymust be engineered. In addition, in some embodiments, the possibility ofimmunogenicity resulting from the pI variants is significantly reducedby importing pI variants from different IgG isotypes such that pI ischanged without introducing significant immunogenicity. Thus, anadditional problem to be solved is the elucidation of low pI constantdomains with high human sequence content, e.g., the minimization oravoidance of non-human residues at any particular position.Alternatively or in addition to isotypic substitutions, the possibilityof immunogenicity resulting from the pI variants is significantlyreduced by utilizing isosteric substitutions (e.g. Asn to Asp; and Glnto Glu).

As discussed below, a side benefit that can occur with this pIengineering is also the extension of serum half-life and increased FcRnbinding. That is, as described in US Publ. App. No. US 2012/0028304(incorporated by reference in its entirety), lowering the pI of antibodyconstant domains (including those found in antibodies and Fc fusions)can lead to longer serum retention in vivo. These pI variants forincreased serum half-life also facilitate pI changes for purification.

In addition, it should be noted that the pI variants give an additionalbenefit for the analytics and quality control process of bispecificantibodies, as the ability to either eliminate, minimize and distinguishwhen homodimers are present is significant. Similarly, the ability toreliably test the reproducibility of the heterodimeric antibodyproduction is important.

In general, embodiments of particular use rely on sets of variants thatinclude skew variants, which encourage heterodimerization formation overhomodimerization formation, coupled with pI variants, which increase thepI difference between the two monomers to facilitate purification ofheterodimers away from homodimers.

Exemplary combinations of pI variants are shown in FIGS. 4 and 5, andFIG. 30 of US Publ. App. No. 2016/0355608, all of which are hereinincorporated by reference in its entirety and specifically for thedisclosure of pI variants. Preferred combinations of pI variants areshown in FIGS. 1 and 2. As outlined herein and shown in the figures,these changes are shown relative to IgG1, but all isotypes can bealtered this way, as well as isotype hybrids. In the case where theheavy chain constant domain is from IgG2-4, R133E and R133Q can also beused.

In one embodiment, a preferred combination of pI variants has onemonomer (the negative Fab side) comprising 208D/295E/384D/418E/421Dvariants (N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) anda second monomer (the positive scFv side) comprising a positivelycharged scFv linker, including (GKPGS)₄ (SEQ ID NO: 1). However, as willbe appreciated by those in the art, the first monomer includes a CH1domain, including position 208. Accordingly, in constructs that do notinclude a CH1 domain (for example for antibodies that do not utilize aCH1 domain on one of the domains), a preferred negative pI variant Fcset includes 295E/384D/418E/421D variants (Q295E/N384D/Q418E/N421D whenrelative to human IgG1).

Accordingly, in some embodiments, one monomer has a set of substitutionsfrom FIG. 2 and the other monomer has a charged linker (either in theform of a charged scFv linker because that monomer comprises an scFv ora charged domain linker, as the format dictates, which can be selectedfrom those depicted in FIG. 5).

In some embodiments, modifications are made in the hinge of the Fcdomain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pImutations and particularly substitutions can be made in one or more ofpositions 216-230, with 1, 2, 3, 4 or 5 mutations finding use. Again,all possible combinations are contemplated, alone or with other pIvariants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327,334 and 339, based on EU numbering. It should be noted that changes in233-236 can be made to increase effector function (along with 327A) inthe IgG2 backbone. Again, all possible combinations of these 14positions can be made; e.g., =may include a variant Fc domain with 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, anon-native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non-native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the modifications can be independently andoptionally selected from position 355, 359, 362, 384, 389, 392, 397,418, 419, 444 and 447 (EU numbering) of the CH3 region. Specificsubstitutions that find use in lowering the pI of CH3 domains include,but are not limited to, a non-native glutamine or glutamic acid atposition 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non-native glutamic acid at position 419, a non-nativeglutamic acid at position 359, a non-native glutamic acid at position362, a non-native glutamic acid at position 389, a non-native glutamicacid at position 418, a non-native glutamic acid at position 444, and adeletion or non-native aspartic acid at position 447.

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 4. As outlinedherein and shown in the figures, these changes are shown relative toIgG1, but all isotypes can be altered this way, as well as isotypehybrids. In the case where the heavy chain constant domain is fromIgG2-4, R133E and R133Q can also be used.

In one embodiment, for example in the FIGS. 21A and 21B formats, apreferred combination of pI variants has one monomer (the negative Fabside) comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a secondmonomer (the positive scFv side) comprising a positively charged scFvlinker, including (GKPGS)₄ (SEQ ID NO: 1). However, as will beappreciated by those in the art, the first monomer includes a CH1domain, including position 208. Accordingly, in constructs that do notinclude a CH1 domain (for example for antibodies that do not utilize aCH1 domain on one of the domains, for example in a dual scFv format or a“one armed” format such as those depicted in FIG. 42B, C or D), apreferred negative pI variant Fc set includes 295E/384D/418E/421Dvariants (Q295E/N384D/Q418E/N421D when relative to human IgG1).

Accordingly, in some embodiments, one monomer has a set of substitutionsfrom FIG. 4 and the other monomer has a charged linker (either in theform of a charged scFv linker because that monomer comprises an scFv ora charged domain linker, as the format dictates, which can be selectedfrom those depicted in FIG. 5).

3. Isotypic Variants

In addition, many embodiments of the antibodies described herein rely onthe “importation” of pI amino acids at particular positions from one IgGisotype into another, thus reducing or eliminating the possibility ofunwanted immunogenicity being introduced into the variants. A number ofthese are shown in FIG. 21 of US Publ. 2014/0370013, hereby incorporatedby reference. That is, IgG1 is a common isotype for therapeuticantibodies for a variety of reasons, including high effector function.However, the heavy constant region of IgG1 has a higher pI than that ofIgG2 (8.10 versus 7.31). By introducing IgG2 residues at particularpositions into the IgG1 backbone, the pI of the resulting monomer islowered (or increased) and additionally exhibits longer serum half-life.For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 hasa glutamic acid (pI 3.22); importing the glutamic acid will affect thepI of the resulting protein. As is described below, a number of aminoacid substitutions are generally required to significant affect the pIof the variant antibody. However, it should be noted as discussed belowthat even changes in IgG2 molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

4. Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Pub.2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of the Fv and scaffold regions.Alternatively, the pI of each monomer can be compared.

5. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall'Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half-life as wild-type Fc (Dall'Acqua et al.2002, J. Immunol. 169:5171-5180, entirely incorporated by reference).The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid therelease of the Fc back into the blood. Therefore, the Fc mutations thatwill increase Fc's half-life in vivo will ideally increase FcRn bindingat the lower pH while still allowing release of Fc at higher pH. Theamino acid histidine changes its charge state in the pH range of 6.0 to7.4. Therefore, it is not surprising to find His residues at importantpositions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

C. Additional Fc Variants for Additional Functionality

In addition to the heterodimerization variants discussed above, thereare a number of useful Fc amino acid modification that can be made for avariety of reasons, including, but not limited to, altering binding toone or more FcγR receptors, altered binding to FcRn receptors, etc, asdiscussed below.

Accordingly, the antibodies provided herein (heterodimeric, as well ashomodimeric) can include such amino acid modifications with or withoutthe heterodimerization variants outlined herein (e.g., the pI variantsand steric variants). Each set of variants can be independently andoptionally included or excluded from any particular heterodimericprotein.

1. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors. In certainembodiments, the subject antibody includes modifications that alter thebinding to one or more FcγR receptors (i.e., “FcγR variants”).Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the antibodies described herein include those listed in U.S.Pat. No. 8,188,321 (particularly FIGS. 41) and U.S. Pat. No. 8,084,582,and US Publ. App. Nos. 20060235208 and 20070148170, all of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein. Particular variantsthat find use include, but are not limited to, 236A, 239D, 239E, 332E,332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,239D/332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half-life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 2591, 428L/434S, 2591/308F, 4361/428L, 4361 or V/434S,436V/428L and 2591/308F/428L. Such modification may be included in oneor both Fc domains of the subject antibody.

2. Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies that bindCD3 monovalently it is generally desirable to ablate FcγRIIIa binding toeliminate or significantly reduce ADCC activity. wherein one of the Fcdomains comprises one or more Fcγ receptor ablation variants. Theseablation variants are depicted in FIG. 14, and each can be independentlyand optionally included or excluded, with preferred aspects utilizingablation variants selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. Itshould be noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

As is known in the art, the Fc domain of human IgG1 has the highestbinding to the Fcγ receptors, and thus ablation variants can be usedwhen the constant domain (or Fc domain) in the backbone of theheterodimeric antibody is IgG1. Alternatively, or in addition toablation variants in an IgG1 background, mutations at the glycosylationposition 297 (generally to A or S) can significantly ablate binding toFcγRIIIa, for example. Human IgG2 and IgG4 have naturally reducedbinding to the Fcγ receptors, and thus those backbones can be used withor without the ablation variants.

D. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In some embodiments, theheterodimeric antibodies provided herein include the combination ofheterodimerization skew variants, isosteric pI substitutions and FcKOvariants as depicted in FIG. 4. In addition, all of these variants canbe combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

Exemplary combination of variants that are included in some embodimentsof the heterodimeric 1+1 Fab-scFv-Fc and 2+1 Fab₂-scFv-Fc formatantibodies are included in FIG. 4. In certain embodiments, the antibodyis a heterodimeric 1+1 Fab-scFv-Fc or 2+1 Fab₂-scFv-Fc format antibodyas shown in FIGS. 21A and 21B.

E. Anti-PSMA×Anti-CD3 Bispecific Antibodies

In another aspect, provided herein are anti-PSMA×anti-CD3 (also referredto herein as “αPSMA×αCD3”) bispecific antibodies. Such antibodiesinclude at least one PSMA binding domain and at least one CD3 bindingdomain. In some embodiments, bispecific αPSMA×αCD3 provided hereinimmune responses selectively in tumor sites that express PSMA.

Note that unless specified herein, the order of the antigen list in thename does not confer structure; that is a PSMA×CD3 1+1 Fab-scFv-Fcantibody can have the scFv bind to PSMA or CD3, although in some cases,the order specifies structure as indicated.

As is more fully outlined herein, these combinations of ABDs can be in avariety of formats, as outlined below, generally in combinations whereone ABD is in a Fab format and the other is in an scFv format. Exemplaryformats that are used in the bispecific antibodies provided hereininclude the 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fv formats (see, e.g.,FIGS. 15A and 15B). Other useful antibody formats include, but are notlimited to, “mAb-Fv,” “mAb-scFv,” “central-Fv”, “one armed scFv-mAb,”“scFv-mAb,” “dual scFv,” and “trident” format antibodies, as disclosedin FIG. 50A-50K.

In addition, in general, one of the ABDs comprises a scFv as outlinedherein, in an orientation from N- to C-terminus of VH-scFv linker-VL orVL-scFv linker-VH. One or both of the other ABDs, according to theformat, generally is a Fab, comprising a VH domain on one protein chain(generally as a component of a heavy chain) and a VL on another proteinchain (generally as a component of a light chain).

As will be appreciated by those in the art, any set of 6 CDRs or VH andVL domains can be in the scFv format or in the Fab format, which is thenadded to the heavy and light constant domains, where the heavy constantdomains comprise variants (including within the CH1 domain as well asthe Fc domain). The scFv sequences contained in the sequence listingutilize a particular charged linker, but as outlined herein, unchargedor other charged linkers can be used, including those depicted in FIG. 5and FIG. 6.

In addition, as discussed above, the numbering used in the SequenceListing for the identification of the CDRs is Kabat, however, differentnumbering can be used, which will change the amino acid sequences of theCDRs as shown in Table 2.

For all of the variable heavy and light domains listed herein, furthervariants can be made. As outlined herein, in some embodiments the set of6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (withamino acid substitutions finding particular use), as well as changes inthe framework regions of the variable heavy and light domains, as longas the frameworks (excluding the CDRs) retain at least about 80, 85 or90% identity to a human germline sequence selected from those listed inFIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend isincorporated by reference in its entirety herein. Thus, for example, theidentical CDRs as described herein can be combined with differentframework sequences from human germline sequences, as long as theframework regions retain at least 80, 85 or 90% identity to a humangermline sequence selected from those listed in FIG. 1 of U.S. Pat. No.7,657,380. Alternatively, the CDRs can have amino acid modifications(e.g., from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs(that is, the CDRs can be modified as long as the total number ofchanges in the set of 6 CDRs is less than 6 amino acid modifications,with any combination of CDRs being changed; e.g., there may be onechange in vlCDR1, two in vhCDR2, none in vhCDR3, etc.)), as well ashaving framework region changes, as long as the framework regions retainat least 80, 85 or 90% identity to a human germline sequence selectedfrom those listed in FIG. 1 of U.S. Pat. No. 7,657,380.

The anti-PSMA×anti-CD3 bispecific antibody can include any suitable CD3ABD, including those described herein (see, e.g., FIGS. 10A-10F). Insome embodiments, the CD3 ABD of the anti-PSMA×anti-CD3 bispecificantibody includes the variable heavy domain and variable light domain ofa CD3 ABD provided herein, including those described in FIGS. 10A-10Fand the sequence listing. In some embodiments, the CD3 ABD includes thevariable heavy domain and variable light domain of one of the followingCD3 ABDs: H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47,H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32,L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F).In exemplary embodiments, the CD3 ABD is one of the following CD3 ABDs:H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47,H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89,L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F) or a variantthereof. The anti-PSMA×anti-CD3 bispecific antibody can include anysuitable PSMA ABD, including those described herein (see, e.g., FIGS.12, 13A-13B, and 14A-14I). In some embodiments, the PSMA ABD of theanti-PSMA×anti-CD3 bispecific antibody includes the variable heavydomain and variable light domain of a PSMA ABD provided herein,including those described in FIGS. 12, 13A-13B, and 14A-14I and thesequence listing. In some embodiments, the PSMA ABD includes thevariable heavy domain and variable light domain of one of the followingPSMA ABDs: PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). In exemplaryembodiments, the PSMA ABD is one of the following PSMA ABDs: PSMA ABDs:PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E) or variants thereof.

F. Useful Formats

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric bispecific antibodies provided herein can takeon a wide variety of configurations, as are generally depicted inFIG. 1. Some figures depict “single ended” configurations, where thereis one type of specificity on one “arm” of the molecule and a differentspecificity on the other “arm”. Other figures depict “dual ended”configurations, where there is at least one type of specificity at the“top” of the molecule and one or more different specificities at the“bottom” of the molecule. Thus, in some embodiments, the antibodiesdescribed herein are directed to novel immunoglobulin compositions thatco-engage a different first and a second antigen.

As will be appreciated by those in the art, the heterodimeric formats ofthe antibodies described herein can have different valencies as well asbe bispecific. That is, heterodimeric antibodies of the antibodiesdescribed herein can be bivalent and bispecific, wherein one targettumor antigen (e.g. CD3) is bound by one binding domain and the othertarget tumor antigen (e.g. PSMA) is bound by a second binding domain.The heterodimeric antibodies can also be trivalent and bispecific,wherein the first antigen is bound by two binding domains and the secondantigen by a second binding domain. As is outlined herein, when CD3 isone of the target antigens, it is preferable that the CD3 is bound onlymonovalently, to reduce potential side effects.

The antibodies described herein utilize anti-CD3 antigen binding domainsin combination with anti-PSMA binding domains. As will be appreciated bythose in the art, any collection of anti-CD3 CDRs, anti-CD3 variablelight and variable heavy domains, Fabs and scFvs as depicted in any ofthe Figures can be used. Similarly, any of the anti-PSMA antigen bindingdomains can be used, whether CDRs, variable light and variable heavydomains, Fabs and scFvs as depicted in any of the Figures (e.g., FIGS.17, 18A-E and 19A-X) can be used, optionally and independently combinedin any combination.

1. 1+1 Fab-scFv-Fc Format

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the “1+1 Fab-scFv-Fc” or “bottle-opener” format asshown in FIG. 21A with an exemplary combination of a CD3 binding domainand a tumor target antigen (PSMA) binding domain. In this embodiment,one heavy chain monomer of the antibody contains a single chain Fv(“scFv”, as defined below) and an Fc domain. The scFv includes avariable heavy domain (VH1) and a variable light domain (VL1), whereinthe VH1 is attached to the VL1 using an scFv linker that can be charged(see, e.g., FIG. 5). The scFv is attached to the heavy chain using adomain linker (see, e.g., FIG. 6). The other heavy chain monomer is a“regular” heavy chain (VH-CH1-hinge-CH2-CH3). The 1+1 Fab-scFv-Fc alsoincludes a light chain that interacts with the VH-CH1 to form a Fab.This structure is sometimes referred to herein as the “bottle-opener”format, due to a rough visual similarity to a bottle-opener. The twoheavy chain monomers are brought together by the use of amino acidvariants (e.g., heterodimerization variants, discussed above) in theconstant regions (e.g., the Fc domain, the CH1 domain and/or the hingeregion) that promote the formation of heterodimeric antibodies as isdescribed more fully below.

There are several distinct advantages to the present “1+1 Fab-scFv-Fc”format. As is known in the art, antibody analogs relying on two scFvconstructs often have stability and aggregation problems, which can bealleviated in the antibodies described herein by the addition of a“regular” heavy and light chain pairing. In addition, as opposed toformats that rely on two heavy chains and two light chains, there is noissue with the incorrect pairing of heavy and light chains (e.g. heavy 1pairing with light 2, etc.).

Many of the embodiments outlined herein rely in general on the 1+1Fab-scFv-Fc or “bottle opener” format antibody that comprises a firstmonomer comprising an scFv, comprising a variable heavy and a variablelight domain, covalently attached using an scFv linker (charged, in manybut not all instances), where the scFv is covalently attached to theN-terminus of a first Fc domain usually through a domain linker (i.e.,from N- to C-terminus scFv-linker-CH2-C3). In some embodiments, thevariable light domain of the scFv is attached to the first Fc domain. Inother embodiments, the variable heavy domain of the scFv is attached tothe first Fc domain. The domain linker can be either charged oruncharged and exogenous or endogenous (e.g., all or part of the nativehinge domain). Any suitable linker can be used to attach the scFv to theN-terminus of the first Fc domain. In some embodiments, the domainlinker is chosen from the domain linkers in FIG. 6. The second monomerof the 1+1 Fab-scFv-Fc format or “bottle opener” format is a heavy chain(i.e., from N- to C-terminus VH-CH1-hinge-CH2-CH3), and the compositionfurther comprises a light chain.

In addition, the Fc domains of the antibodies described herein generallyinclude skew variants (e.g. a set of amino acid substitutions as shownin FIGS. 1 and 4, with particularly useful skew variants being selectedfrom the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L;K370S:S364K/E357Q; T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In general, in many preferred embodiments, the scFv is the domain thatbinds to the CD3, and the Fab forms a PSMA binding domain. An exemplaryanti-PSMA×anti-CD3 bispecific antibody in the 1+1 Fab-scFv-Fc format isdepicted in FIG. 21A. Exemplary anti-PSMA×anti-CD3 bispecific antibodyin the 1+1 Fab-scFv-Fc format is depicted in FIGS. 22-25. Exemplaryvariable heavy and light domains of the scFv that binds to CD3 areincluded in FIGS. 10A-10F. Exemplary variable heavy and light domains ofthe Fv that binds to PSMA are included in FIGS. 17 and 18. In anexemplary embodiment, the PSMA binding domain of the 1+1 Fab-scFv-FcPSMA×CD3 bispecific antibody includes the VH of PSMA-H H1 (FIG. 17) andVL of one of the following PSMA binding domains: PSMA-H L1 andL1.1-L1.84 (FIGS. 17 and 18A-18E). In one embodiment, the CD3 bindingdomain of the 1+1 Fab-scFv-Fc PSMA×CD3 bispecific antibody includes theVH and VL of one of the following CD3 binding domains: H1.30_L1.47,H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47,L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90,L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F). Particularly useful PSMAand CD3 combinations for use in the 1+1 Fab-scFv-Fc PSMA×CD3 bispecificantibody format are disclosed in FIGS. 22-25 and include: a) CD3H1.30_L1.47×PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; or PSMA-H H1_L1.13; b) CD3 H1.31_L1.47, CD3 H1.32_L1.47, orCD3 H1.33_L1.47×PSMA-H H1L1.

In certain embodiments, the 1+1 Fab-scFv-Fc scaffold format includes afirst monomer that includes a scFv-domain linker-CH2-CH3 monomer, asecond monomer that includes a first variable heavydomain-CH1-hinge-CH2-CH3 monomer and a third monomer that includes afirst variable light domain. In some embodiments, the CH2-CH3 of thefirst monomer is a first variant Fc domain and the CH2-CH3 of the secondmonomer is a second variant Fc domain. In some embodiments, the scFvincludes a scFv variable heavy domain and a scFv variable light domainthat form a CD3 binding moiety. In certain embodiments, the scFvvariable heavy domain and scFv variable light domain are covalentlyattached using an scFv linker (charged, in many but not all instances.See, e.g., FIG. 5). In some embodiments, the first variable heavy domainand first variable light domain form a PSMA binding domain. CD3 bindingdomain sequences finding particular use in these embodiments include,but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47,H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30,L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (seeFIGS. 10A-10F). PSMA binding domain sequences that are of particular usein these embodiments include, but are not limited to, PSMA-H H1_L1,PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26;PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52;PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-H H1_L1.84; and PSMA-H H1_L1.13.Particularly useful PSMA and CD3 combinations for use in the 1+1Fab-scFv-Fc PSMA×CD3 bispecific antibody format are disclosed in FIGS.22-25 and include: a) CD3 H1.30_L1.47×PSMA-H H1_L1, PSMA-H H1_L1.58;PSMA-H H1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75;PSMA-H H1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78;PSMA-H H1_L1.81; PSMA-H H1_L1.84; or PSMA-H H1_L1.13; b) CD3H1.31_L1.47, CD3 H1.32_L1.47, or CD3 H1.33_L1.47×PSMA-H H1L1.

In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants,pI variants, and ablation variants. Accordingly, some embodimentsinclude 1+1 Fab-scFv-Fc formats that comprise: a) a first monomer (the“scFv monomer”) that comprises a charged scFv linker (with the +Hsequence of FIG. 5 being preferred in some embodiments), the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and an scFv that binds to CD3 asoutlined herein; b) a second monomer (the “Fab monomer”) that comprisesthe skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain; and c) alight chain that includes a variable light domain light domain (VL) anda constant light domain (CL), wherein numbering is according to EUnumbering. The variable heavy domain and variable light domain make up aPSMA binding moiety. CD3 binding domain sequences finding particular usein these embodiments include, but are not limited to, H1.30_L1.47,H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47,L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90,L1.47_H1.33, and L1.47_H1.31 (see FIGS. 10A-10F). PSMA binding domainsequences that are of particular use in these embodiments include, butare not limited to, PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11;PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68;PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81;PSMA-H H1_L1.84; and PSMA-H H1_L1.13. Particularly useful PSMA and CD3combinations for use in the 1+1 Fab-scFv-Fc PSMA×CD3 bispecific antibodyformat are disclosed in FIGS. 22-25 and include: a) CD3H1.30_L1.47×PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; or PSMA-H H1_L1.13; b) CD3 H1.31_L1.47, CD3 H1.32_L1.47, orCD3 H1.33_L1.47×PSMA-H H1L1.

In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants,pI variants, ablation variants and FcRn variants. Accordingly, someembodiments include 1+1 Fab-scFv-Fc formats that comprise: a) a firstmonomer (the “scFv monomer”) that comprises a charged scFv linker (withthe +H sequence of FIG. 6 being preferred in some embodiments), the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and anscFv that binds to CD3 as outlined herein; b) a second monomer (the “Fabmonomer”) that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S, and avariable heavy domain; and c) a light chain that includes a variablelight domain (VL) and a constant light domain (CL), wherein numbering isaccording to EU numbering. The variable heavy domain and variable lightdomain make up a PSMA binding domain. CD3 binding domain sequencesfinding particular use in these embodiments include, but are not limitedto, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47,H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89,L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (see FIGS. 10A-10F). PSMAbinding domain sequences that are of particular use in these embodimentsinclude, but are not limited to, PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-HH1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-HH1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-HH1_L1.81; PSMA-H H1_L1.84; and PSMA-H H1_L1.13. Particularly useful PSMAand CD3 combinations for use in the 1+1 Fab-scFv-Fc PSMA×CD3 bispecificantibody format are disclosed in FIGS. 22-25 and include: a) CD3H1.30_L1.47×PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; or PSMA-H H1_L1.13; b) CD3 H1.31_L1.47, CD3 H1.32_L1.47, orCD3 H1.33_L1.47×PSMA-H H1L1.

FIGS. 7A-7D show some exemplary Fc domain sequences that are useful inthe 1+1 Fab-scFv-Fc format antibodies. The “monomer 1” sequencesdepicted in FIGS. 7A-7D typically refer to the Fc domain of the “Fab-Fcheavy chain” and the “monomer 2” sequences refer to the Fc domain of the“scFv-Fc heavy chain.” Further, FIG. 9 provides useful CL sequences thatcan be used with this format. In some embodiments, any of the VH and VLsequences depicted herein (including all VH and VL sequences depicted inthe Figures and Sequence Listings, including those directed to PSMA) canbe added to the bottle opener backbone formats of FIG. 7A-7D as the “Fabside”, using any of the anti-CD3 scFv sequences shown in the Figures andSequence Listings. For bottle opener backbone 1 from FIG. 7A,(optionally including the 428L/434S variants), CD binding domainsequences finding particular use in these embodiments include, but arenot limited to, CD3 binding domain anti-CD3 H1.30_L1.47, anti-CD3H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3H1.33_L1.47 and anti-CD3 H1.31_L1.47, as well as those depicted in FIGS.10A-10F, attached as the scFv side of the backbones shown in FIGS.7A-7D. Particularly useful PSMA and CD3 sequence combinations(optionally including the 428L/434S variants) and exemplaryanti-CD3×anti-PSMA antibodies in the 1+1 Fab-scFv-Fc format are depictedin FIGS. 22-25.

2. mAb-Fv

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the mAb-Fv format (FIG. 50G). In this embodiment,the format relies on the use of a C-terminal attachment of an “extra”variable heavy domain to one monomer and the C-terminal attachment of an“extra” variable light domain to the other monomer, thus forming a thirdantigen binding domain, wherein the Fab portions of the two monomersbind a PSMA and the “extra” scFv domain binds CD3.

In this embodiment, the first monomer comprises a first heavy chain,comprising a first variable heavy domain and a first constant heavydomain comprising a first Fc domain, with a first variable light domaincovalently attached to the C-terminus of the first Fc domain using adomain linker (VH1-CH1-hinge-CH2-CH3-[optional linker]-VL2). The secondmonomer comprises a second variable heavy domain of the second constantheavy domain comprising a second Fc domain, and a third variable heavydomain covalently attached to the C-terminus of the second Fc domainusing a domain linker (vh1-CH1-hinge-CH2-CH3-[optional linker]-VH2. Thetwo C-terminally attached variable domains make up a Fv that binds CD3(as it is less preferred to have bivalent CD3 binding). This embodimentfurther utilizes a common light chain comprising a variable light domainand a constant light domain that associates with the heavy chains toform two identical Fabs that bind a PSMA. As for many of the embodimentsherein, these constructs include skew variants, pI variants, ablationvariants, additional Fc variants, etc. as desired and described herein.

The antibodies described herein provide mAb-Fv formats where the CD3binding domain sequences are as shown in FIGS. 10A-10F. The antibodiesdescribed herein provide mAb-Fv formats wherein the PSMA binding domainsequences are as shown in FIGS. 17 and 18.

In addition, the Fc domains of the mAb-Fv format comprise skew variants(e.g. a set of amino acid substitutions as shown in FIGS. 1 and 4, withparticularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includemAb-Fv formats that comprise: a) a first monomer that comprises the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to PSMA, and a second variable heavy domain; b) a secondmonomer that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain, makes up the Fv that binds to PSMAas outlined herein, and a second variable light chain, that togetherwith the second variable heavy domain forms an Fv (ABD) that binds toCD3; and c) a light chain comprising a first variable light domain and aconstant light domain.

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-Fv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domainof the light chain, makes up an Fv that binds to PSMA, and a secondvariable heavy domain; b) a second monomer that comprises the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variantsM428L/N434S and a first variable heavy domain that, with the firstvariable light domain, makes up the Fv that binds to PSMA as outlinedherein, and a second variable light chain, that together with the secondvariable heavy domain of the first monomer forms an Fv (ABD) that bindsCD3; and c) a light chain comprising a first variable light domain and aconstant light domain.

3. mAb-scFv

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the mAb-scFv format (FIG. 50H). In this embodiment,the format relies on the use of a C-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers bind PSMA and the “extra” scFvdomain binds CD3. Thus, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aC-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation (VH1-CH1-hinge-CH2-CH3-[optional linker]-VH2-scFv linker-VL2or VH1-CH1-hinge-CH2-CH3-[optional linker]-VL2-scFv linker-VH2). Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain, that associates with the heavychains to form two identical Fabs that bind PSMA. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The antibodies described herein provide mAb-scFv formats where the CDbinding domain sequences are as shown in FIGS. 10A-10F and the PSMAbinding domain sequences are as shown in FIGS. 17 and 18A-18E.

In addition, the Fc domains of the mAb-scFv format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIG. 1,with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includemAb-scFv formats that comprise: a) a first monomer that comprises theskew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein, and a scFv domain that binds to CD3;b) a second monomer that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein; and c) a common light chain comprisinga variable light domain and a constant light domain.

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-scFv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to PSMA as outlined herein, and ascFv domain that binds to CD3; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to PSMA as outlined herein; andc) a common light chain comprising a variable light domain and aconstant light domain.

4. 2+1 Fab₂-scFv-Fc Format

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the “2+1 Fab₂-scFv-Fc” format (also referred to inprevious related filings as “central-scFv format”) shown in FIG. 21Bwith an exemplary combination of a CD3 binding domain and two tumortarget antigen (PSMA) binding domains. In this embodiment, the formatrelies on the use of an inserted scFv domain thus forming a thirdantigen binding domain, wherein the Fab portions of the two monomersbind PSMA and the “extra” scFv domain binds CD3. The scFv domain isinserted between the Fc domain and the CH1-Fv region of one of themonomers, thus providing a third antigen binding domain. As described,PSMA×CD3 bispecific antibodies having the 2+1 Fab₂-scFv-Fc format arepotent in inducing redirected T cell cytotoxicity in cellularenvironments that express low levels of PSMA. Moreover, as shown in theexamples, PSMA×CD3 bispecific antibodies having the 2+1 Fab2-scFv-Fcformat allow for the “fine tuning” of immune responses as suchantibodies exhibit a wide variety of different properties, depending onthe PSMA and/or CD3 binding domains used. For example, such antibodiesexhibit differences in selectivity for cells with different PSMAexpression, potencies for PSMA expressing cells, ability to elicitcytokine release, and sensitivity to soluble PSMA. These PSMA antibodiesfind use, for example, in the treatment of PSMA associated cancers.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain (and optional hinge) and Fcdomain, with a scFv comprising a scFv variable light domain, an scFvlinker and a scFv variable heavy domain. The scFv is covalently attachedbetween the C-terminus of the CH1 domain of the heavy constant domainand the N-terminus of the first Fc domain using optional domain linkers(N- to C-terminus: VH1-CH1-[optionallinker]-VH2-scFvlinker-VL2-[optional linker including thehinge]-CH2-CH3, or the opposite orientation for the scFv, N- toC-terminus: VH1-CH1-[optional linker]-VL2-scFvlinker-VH2-[optionallinker including the hinge]-CH2-CH3). The optional linkers can be anysuitable peptide linkers, including, for example, the domain linkersincluded in FIG. 6. In some embodiments, the optional linker is a hingeor a fragment thereof. The other monomer is a standard Fab side (i.e.,VH1-CH1-hinge-CH2-CH3). This embodiment further utilizes a common lightchain comprising a variable light domain and a constant light domainthat associates with the heavy chains to form two identical Fabs thatbind PSMA. As for many of the embodiments herein, these constructsinclude skew variants, pI variants, ablation variants, additional Fcvariants, etc. as desired and described herein.

In one embodiment, the 2+1 Fab₂-scFv-Fc format antibody includes an scFvwith the VH and VL of a CD3 binding domain sequence depicted in FIGS.10A-10F or the Sequence Listing. In one embodiment, the 2+1 Fab₂-scFv-Fcformat antibody includes two Fabs having the VH and VL of a PSMA bindingdomain as shown in FIGS. 17 and 18A-18E and the Sequence Listing. In anexemplary embodiment, the PSMA binding domain of the 2+1 Fab₂-scFv-FcPSMA×CD3 bispecific antibody includes the V_(H) of PSMA-H H1 (FIG. 17)and VL of one of the following PSMA binding domains: PSMA-H L1 andL1.1-L1.84 (FIGS. 17 and 18A-18E). In one embodiment, the CD3 bindingdomain of the 2+1 Fab2-scFv-Fc format antibody includes the VH and VL ofone of the following CD3 binding domains: H1.30_L1.47, H1.32_L1.47,H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30,L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, andL1.47_H1.31 (FIGS. 10A-10F). Particularly useful PSMA and CD3combinations for use in the 2+1 Fab₂-scFv-Fc format antibody format aredisclosed in FIGS. 26-32 and include: a) CD3 H1.30_L1.47, CD3H1.32_L1.47, CD3 L1.47_H1.32, CD3 H1.89_L1.47, CD3 L1.47_H1.89, CD3H1.33_L1.47, CD3 L1.47_H1.32×b) PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-HH1_L1.11; PSMA-H H1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-HH1_L1.68; PSMA-H H1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-HH1_L1.81; PSMA-H H1_L1.84

In addition, the Fc domains of the 2+1 Fab₂-scFv-Fc format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIG. 1,with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the 2+1 Fab₂-scFv-Fc format antibody includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments include 2+1 Fab₂-scFv-Fc formats that comprise: a) a firstmonomer (the Fab-scFv-Fc side) that comprises the skew variantsS364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, anda variable heavy domain that, with the variable light domain of thecommon light chain, makes up an Fv that binds to PSMA as outlinedherein, and an scFv domain that binds to CD3; b) a second monomer (theFab-Fc side) that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withvariable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein; and c) a common light chain comprisingthe variable light domain and a constant light domain, where numberingis according to EU numbering. In some embodiments, the common lightchain and variable heavy domains on each monomer for PSMA bindingdomains. CD3 binding domain sequences finding particular use in theseembodiments include, but are not limited to, H1.30_L1.47, H1.32_L1.47,H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30,L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, andL1.47_H1.31 as well as those depicted in FIGS. 10A-10F. PSMA bindingdomain sequences that are of particular use in these embodimentsinclude, but are not limited to, PSMA-H H1_L1, and PSMA-H H1_L1.1-L1.84as depicted in FIGS. 17-19.

In some embodiments, the 2+1 Fab₂-scFv-Fc format antibody includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments include 2+1 Fab₂-scFv-Fc formats that comprise: a) afirst monomer (the Fab-scFv-Fc side) that comprises the skew variantsS364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, theFcRn variants M428L/N434S and a variable heavy domain that, with thevariable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein, and an scFv domain that binds to CD3;b) a second monomer (the Fab-Fc side) that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a variable heavy domain that, with variable light domain of thecommon light chain, makes up an Fv that binds to PSMA as outlinedherein; and c) a common light chain comprising a variable light domainand a constant light domain, where numbering is according to EUnumbering. In some embodiments, the common light chain and variableheavy domains on each monomer for PSMA binding domains. CD3 bindingdomain sequences finding particular use in these embodiments include,but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47,H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30,L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 aswell as those depicted in FIGS. 10A-10F. PSMA binding domain sequencesthat are of particular use in these embodiments include, but are notlimited to, PSMA-H H1_L1, and PSMA-H H1_L1.1-L1.84 as depicted in FIGS.17-19.

FIGS. 8A-8C shows some exemplary Fc domain sequences that are usefulwith the 2+1 Fab₂-scFv-Fc format. The “monomer 1” sequences depicted inFIGS. 8A-8C typically refer to the Fc domain of the “Fab-Fc heavy chain”and the “monomer 2” sequences refer to the Fc domain of the “Fab-scFv-Fcheavy chain.” Further, FIG. 9 provides useful CL sequences that can beused with this format.

Exemplary anti-CD3×anti-PSMA antibodies in the 2+1 Fab₂-scFv-Fc formatare depicted in FIGS. 26-32.

5. Central-Fv

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the central-Fv format (FIG. 50I). In thisembodiment, the format relies on the use of an inserted Fv domain (i.e.,the central Fv domain) thus forming an “extra” third antigen bindingdomain, wherein the Fab portions of the two monomers bind a PSMA and the“extra” central Fv domain binds CD3. The “extra” central Fv domain isinserted between the Fc domain and the CH1-Fv region of the monomers,thus providing a third antigen binding domain (i.e., the “extra” centralFv domain), wherein each monomer contains a component of the “extra”central Fv domain (i.e., one monomer comprises the variable heavy domainand the other a variable light domain of the “extra” central Fv domain).

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain, and Fc domain and anadditional variable light domain. The light domain is covalentlyattached between the C-terminus of the CH1 domain of the heavy constantdomain and the N-terminus of the first Fc domain using domain linkers(VH1-CH1-[optional linker]-VL2-hinge-CH2-CH3). The other monomercomprises a first heavy chain comprising a first variable heavy domain,a CH1 domain and Fc domain and an additional variable heavy domain(VH1-CH1-[optional linker]-VH2-hinge-CH2-CH3). The light domain iscovalently attached between the C-terminus of the CH1 domain of theheavy constant domain and the N-terminus of the first Fc domain usingdomain linkers.

This embodiment further utilizes a common light chain comprising avariable light domain and a constant light domain that associates withthe heavy chains to form two identical Fabs that each bind a PSMA. Asfor many of the embodiments herein, these constructs include skewvariants, pI variants, ablation variants, additional Fc variants, etc.as desired and described herein.

The antibodies described herein provide central-Fv formats where the CD3binding domain sequences are as shown in 10A-10F and the PSMA bindingdomain sequences (VH, VL and CDRs) are as shown in FIGS. 17 and 18.

6. One Armed Central-scFv

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the one armed central-scFv format (FIG. 50C). Inthis embodiment, one monomer comprises just an Fc domain, while theother monomer includes a Fab domain (a first antigen binding domain), ascFv domain (a second antigen binding domain) and an Fc domain, wherethe scFv domain is inserted between the Fc domain and the Fc domain. Inthis format, the Fab portion binds one receptor target and the scFvbinds another. In this format, either the Fab portion binds a PSMA andthe scFv binds CD3 or vice versa.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain, with a scFvcomprising a scFv variable light domain, an scFv linker and a scFvvariable heavy domain. The scFv is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers, in eitherorientation, VH1-CH1-[optional domain linker]-VH2-scFvlinker-VL2-[optional domain linker]-CH2-CH3 or VH1-CH1-[optional domainlinker]-VL2-scFv linker-VH2-[optional domain linker]-CH2-CH3. The secondmonomer comprises an Fc domain (CH2-CH3). This embodiment furtherutilizes a light chain comprising a variable light domain and a constantlight domain that associates with the heavy chain to form a Fab.

As for many of the embodiments herein, these constructs include skewvariants, pI variants, ablation variants, additional Fc variants, etc.as desired and described herein.

The antibodies described herein provide central-Fv formats where the CD3binding domain sequences are as shown in FIGS. 10A-10F and the PSMAbinding domain sequences (VH, VL and CDRs) are as shown in FIGS. 17 and18.

In addition, the Fc domains of the one armed central-scFv formatgenerally include skew variants (e.g. a set of amino acid substitutionsas shown in FIG. 1, with particularly useful skew variants beingselected from the group consisting of S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments of the one armed central-scFv formats comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a variable heavy domainthat, with the variable light domain of the light chain, makes up an Fvthat binds to PSMA as outlined herein, and a scFv domain that binds toCD3; b) a second monomer that includes an Fc domain having the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments of the one armed central-scFv formats comprise: a) afirst monomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a variable heavy domain that, with the variable light domain of thelight chain, makes up an Fv that binds to PSMA as outlined herein, and ascFv domain that binds to CD3; b) a second monomer that includes an Fcdomain having the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; andc) a light chain comprising a variable light domain and a constant lightdomain.

7. One Armed scFv-mAb

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the one armed scFv-mAb format (FIG. 50D). In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer uses a scFv domain attached at the N-terminus of the heavychain, generally through the use of a linker: VH-scFvlinker-VL-[optional domain linker]-CH1-hinge-CH2-CH3 or (in the oppositeorientation) VL-scFv linker-VH-[optional domainlinker]-CH1-hinge-CH2-CH3. In this format, the Fab portions each bindPSMA and the scFv binds CD3. This embodiment further utilizes a lightchain comprising a variable light domain and a constant light domainthat associates with the heavy chain to form a Fab. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The antibodies described herein provide one armed scFv-mAb formats wherethe CD3 binding domain sequences are as shown in 10A-10F and wherein thePSMA binding domain sequences (VH, VL and CDRs) are as shown in FIGS. 17and 18.

In addition, the Fc domains of the one armed scFv-mAb format generallyinclude skew variants (e.g. a set of amino acid substitutions as shownin FIG. 1, with particularly useful skew variants being selected fromthe group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments of the one armed scFv-mAb formats comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a variable heavy domainthat, with the variable light domain of the light chain, makes up an Fvthat binds to PSMA as outlined herein, and a scFv domain that binds toCD3; b) a second monomer that includes an Fc domain having the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments one armed scFv-mAb formats comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the lightchain, makes up an Fv that binds to PSMA as outlined herein, and a scFvdomain that binds to CD3; b) a second monomer that includes an Fc domainhaving the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; andc) a light chain comprising a variable light domain and a constant lightdomain.

8. scFv-mAb

One heterodimeric scaffold that finds particular use in the antibodiesdescribed herein is the mAb-scFv format (FIG. 50E). In this embodiment,the format relies on the use of a N-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers bind PSMA and the “extra” scFvdomain binds CD3.

In this embodiment, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aN-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation ((VH1-scFv linker-VL1-[optional domainlinker]-VH2-CH1-hinge-CH2-CH3) or (with the scFv in the oppositeorientation) ((VL1-scFv linker-VH1-[optional domainlinker]-VH2-CH1-hinge-CH2-CH3)). This embodiment further utilizes acommon light chain comprising a variable light domain and a constantlight domain that associates with the heavy chains to form two identicalFabs that bind PSMA. As for many of the embodiments herein, theseconstructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein.

The antibodies described herein provide scFv-mAb formats where the CD3binding domain sequences are as shown in 10A-10F and wherein the PSMAbinding domain sequences (VH, VL and CDRs) are as shown in FIGS. 17 and18.

In addition, the Fc domains of the scFv-mAb format generally includeskew variants (e.g. a set of amino acid substitutions as shown in FIG.1, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 3), optionally charged scFv linkers(including those shown in FIG. 5) and the heavy chain comprises pIvariants (including those shown in FIG. 2).

In some embodiments, the scFv-mAb format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includescFv-mAb formats that comprise: a) a first monomer that comprises theskew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein, and a scFv domain that binds to CD3;b) a second monomer that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to PSMA as outlined herein; and c) a common light chain comprisinga variable light domain and a constant light domain.

In some embodiments, the scFv-mAb format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include scFv-mAb formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to PSMA as outlined herein, and ascFv domain that binds to CD3; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to PSMA as outlined herein; andc) a common light chain comprising a variable light domain and aconstant light domain.

9. Dual scFv Formats

The antibodies described herein also provide dual scFv formats (FIG.50B) as are known in the art. In this embodiment, the PSMA×CD3heterodimeric bispecific antibody is made up of two scFv-Fc monomers(both in either (VH-scFv linker-VL-[optional domain linker]-CH2-CH3)format or (VL-scFv linker-VH-[optional domain linker]-CH2-CH3) format,or with one monomer in one orientation and the other in the otherorientation.

The antibodies described herein provide dual scFv formats where the CD3binding domain sequences are as shown in FIGS. 10A-10F and wherein thePSMA binding domain sequences (VH, VL and CDRs) are as shown in FIGS. 17and 18. In some embodiments, the dual scFv format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments include dual scFv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first scFv that binds either CD3or PSMA; and b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a second scFv that bindseither CD3 or PSMA. In some embodiments, the dual scFv format includesskew variants, pI variants, ablation variants and FcRn variants. In someembodiments, the dual scFv format includes skew variants, pI variants,and ablation variants. Accordingly, some embodiments include dual scFvformats that comprise: a) a first monomer that comprises the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst scFv that binds either CD3 or PSMA; and b) a second monomer thatcomprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and asecond scFv that binds either CD3 or PSMA.

10. Non-Heterodimeric Bispecific Antibodies

As will be appreciated by those in the art, the PSMA and CD3 Fvsequences outlined herein can also be used in both monospecificantibodies (e.g., “traditional monoclonal antibodies”) ornon-heterodimeric bispecific formats.

CD3 binding domain sequences finding particular use include, but are notlimited to H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47,H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32,L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 (FIGS. 10A-10F).

PSMA binding domain sequences that are of particular use include, butare not limited to: PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E).

Suitable non-heterodimeric bispecific formats are known in the art, andinclude a number of different formats as generally depicted in Spiess etal., Molecular Immunology (67):95-106 (2015) and Kontermann, mAbs 4:2,182-197 (2012), both of which are expressly incorporated by referenceand in particular for the figures, legends and citations to the formatstherein.

11. Trident Format

In some embodiments, the bispecific antibodies described herein are inthe “Trident” format (FIG. 50K) as generally described in WO2015/184203,hereby expressly incorporated by reference in its entirety and inparticular for the Figures, Legends, definitions and sequences of“Heterodimer-Promoting Domains” or “HPDs”, including “K-coil” and“E-coil” sequences. Tridents rely on using two different HPDs thatassociate to form a heterodimeric structure as a component of thestructure, see FIG. 1K. In this embodiment, the Trident format include a“traditional” heavy and light chain (e.g., VH1-CH1-hinge-CH2-CH3 andVL1-CL), a third chain comprising a first “diabody-type binding domain”or “DART®”, VH2-(linker)-VL3-HPD1 and a fourth chain comprising a secondDART®, VH3-(linker)-(linker)-VL2-HPD2. The VH1 and VL1 form a first ABD,the VH2 and VL2 form a second ABD, and the VH3 and VL3 form a third ABD.In some cases, as is shown in FIG. 1K, the second and third ABDs bindthe same antigen, in this instance generally PSMA, e.g., bivalently,with the first ABD binding a CD3 monovalently.

12. Monospecific, Monoclonal Antibodies

As will be appreciated by those in the art, the novel Fv sequencesoutlined herein can also be used in both monospecific antibodies (e.g.,“traditional monoclonal antibodies”) or non-heterodimeric bispecificformats. Accordingly, in some embodiments, the antibodies describedherein provide monoclonal (monospecific) antibodies comprising the 6CDRs and/or the vh and vl sequences from the figures, generally withIgG1, IgG2, IgG3 or IgG4 constant regions, with IgG1, IgG2 and IgG4(including IgG4 constant regions comprising a S228P amino acidsubstitution) finding particular use in some embodiments. That is, anysequence herein with a “H_L” designation can be linked to the constantregion of a human IgG1 antibody.

In some embodiments, the monospecific antibody is a PSMA monospecificantibody. In certain embodiments, the monospecific anti-PSMA antibodyincludes the 6 CDRs of any of the anti-PSMA binding domains selectedfrom: PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). In certainembodiments, the monospecific anti-PSMA antibody includes the variableheavy domain (VH) and variable light domain (VL) of any of the anti-PSMAbinding domains selected from: PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and18A-18E).

G. Antigen Binding Domains

As discussed herein, the subject heterodimeric antibodies include twoantigen binding domains (ABDs), each of which bind to PSMA or CD3. Asoutlined herein, these heterodimeric antibodies can be bispecific andbivalent (each antigen is bound by a single ABD, for example, in theformat depicted in FIG. 21A), or bispecific and trivalent (one antigenis bound by a single ABD and the other is bound by two ABDs, for exampleas depicted in FIG. 21B).

In addition, in general, one of the ABDs comprises a scFv as outlinedherein, in an orientation from N- to C-terminus of VH-scFv linker-VL orVL-scFv linker-VH. One or both of the other ABDs, according to theformat, generally is a Fab, comprising a VH domain on one protein chain(generally as a component of a heavy chain) and a VL on another proteinchain (generally as a component of a light chain).

The disclosure provides a number of ABDs that bind to a number ofdifferent checkpoint proteins, as outlined below. As will be appreciatedby those in the art, any set of 6 CDRs or VH and VL domains can be inthe scFv format or in the Fab format, which is then added to the heavyand light constant domains, where the heavy constant domains comprisevariants (including within the CH1 domain as well as the Fc domain). ThescFv sequences contained in the sequence listing utilize a particularcharged linker, but as outlined herein, uncharged or other chargedlinkers can be used, including those depicted in FIG. 6.

In addition, as discussed above, the numbering used in the SequenceListing for the identification of the CDRs is Kabat, however, differentnumbering can be used, which will change the amino acid sequences of theCDRs as shown in Table 2.

For all of the variable heavy and light domains listed herein, furthervariants can be made. As outlined herein, in some embodiments the set of6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (withamino acid substitutions finding particular use), as well as changes inthe framework regions of the variable heavy and light domains, as longas the frameworks (excluding the CDRs) retain at least about 80, 85 or90% identity to a human germline sequence selected from those listed inFIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend isincorporated by reference in its entirety herein. Thus, for example, theidentical CDRs as described herein can be combined with differentframework sequences from human germline sequences, as long as theframework regions retain at least 80, 85 or 90% identity to a humangermline sequence selected from those listed in FIG. 1 of U.S. Pat. No.7,657,380. Alternatively, the CDRs can have amino acid modifications(e.g. from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs(that is, the CDRs can be modified as long as the total number ofchanges in the set of 6 CDRs is less than 6 amino acid modifications,with any combination of CDRs being changed; e.g. there may be one changein VLCDR1, two in VHCDR2, none in VHCDR3, etc.)), as well as havingframework region changes, as long as the framework regions retain atleast 80, 85 or 90% identity to a human germline sequence selected fromthose listed in FIG. 1 of U.S. Pat. No. 7,657,380.

1. PSMA Antigen Binding Domains

Provided herein are PSMA antigen binding domain and antibodies thatinclude such binding domains. Suitable sets of 6 CDRs and/or VH and VLdomains included in PSMA binding domains are depicted in FIG. 17(anti-PSMA vhCDRs and VH) and FIGS. 17 and 18A-E (anti-PSMA vlCDRs andVL). In some embodiments, the heterodimeric antibody is a 1+1Fab-scFv-Fc or 2+1 Fab₂-scFv-Fv format antibody (see, e.g., FIGS. 21Aand 21B).

In one embodiment, the PSMA antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of a PSMA ABD described herein, includingthe figures and sequence listing. In certain embodiments the PSMAantigen binding domain includes a variable heavy domain that includesvhCDR1-3 of PSMA-H H1 (FIG. 17) and a variable light domain thatincludes the vlCDR1-3 of any of PSMA-H L1 and L1.1-L1.84 (FIGS. 17 and18A-18E). In exemplary embodiments, the PSMA antigen binding domainincludes the 6 CDRs of one of the following PSMA antigen bindingdomains: PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; and PSMA-H H1_L1.13.

In one embodiment, the PSMA antigen binding domain includes the variableheavy and variable light domain of a PSMA ABD described herein,including the figures and sequence listing. In certain embodiments thePSMA antigen binding domain includes a variable heavy domain that thatis PSMA-H H1 (FIG. 17) and a variable light domain selected from PSMA-HL1 and L1.1-L1.84 (FIGS. 17 and 18A-18E). In exemplary embodiments, thePSMA antigen binding domain includes the variable heavy domain andvariable light domain of one of the following PSMA antigen bindingdomains: PSMA-H H1_L1, PSMA-H H1_L1.58; PSMA-H H1_L1.11; PSMA-HH1_L1.24; PSMA-H H1_L1.26; PSMA-H H1_L1.75; PSMA-H H1_L1.68; PSMA-HH1_L1.29; PSMA-H H1_L1.52; PSMA-H H1_L1.78; PSMA-H H1_L1.81; PSMA-HH1_L1.84; and PSMA-H H1_L1.13.

As will be appreciated by those in the art, suitable PSMA bindingdomains can comprise a set of 6 CDRs as depicted in the Figures, eitheras they are underlined or, in the case where a different numberingscheme is used as described herein and as shown in Table 2, as the CDRsthat are identified using other alignments within the VH and VLsequences of those depicted in FIGS. 17 and 18A-E. Suitable ABDs canalso include the entire VH and VL sequences as depicted in thesesequences and Figures, used as scFvs or as Fabs. In many of theembodiments herein that contain an Fv to PSMA, it is the Fab monomerthat binds PSMA.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to PSMA, the disclosure providesvariant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2, 3, 4or 5 amino acid changes from the parental CDRs, as long as the PSMA ABDis still able to bind to the target antigen, as measured by at least oneof a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to PSMA, the disclosure providesvariant VH and VL domains. In one embodiment, the variant VH and VLdomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental VH and VL domain, as long as the ABD is stillable to bind to the target antigen, as measured at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantVH and VL are at least 90, 95, 97, 98 or 99% identical to the respectiveparental VH or VL, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

2. CD3 Antigen Binding Domains

In some embodiments, one of the ABDs binds CD3. Suitable sets of 6 CDRsand/or V_(H) and V_(L) domains, as well as scFv sequences, are depictedin FIGS. 10A-10F and the Sequence Listing. CD3 binding domain sequencesthat are of particular use include, but are not limited to, anti-CD3H1.30_L1.47, anti-CD3 H1.32, anti-CD3 L1.47, anti-CD3 H1.89_L1.47,anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.47, anti-CD3 H1.31_L1.47,anti-CD3 L1.47_H1.30, anti-CD3 L1.47_H1.30, anti-CD3 L1.47_H1.32,anti-CD3 L1.47_H1.89, anti-CD3 L1.47_H1.90, anti-CD3 L1.47_H1.33, andanti-CD3 L1.47_H1.31 as depicted in FIGS. 10A-10F.

As will be appreciated by those in the art, suitable CD3 binding domainscan comprise a set of 6 CDRs as depicted in FIGS. 10A-10F, either asthey are underlined or, in the case where a different numbering schemeis used as described herein and as shown in Table 2, as the CDRs thatare identified using other alignments within the VH and VL sequences ofthose depicted in FIGS. 10A-10F. Suitable ABDs can also include theentire VH and VL sequences as depicted in these sequences and Figures,used as scFvs or as Fabs. In many of the embodiments herein that containan Fv to CD3, it is the scFv monomer that binds CD3.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to CD3, the disclosure providesvariant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2, 3, 4or 5 amino acid changes from the parental CDRs, as long as the CD3 ABDis still able to bind to the target antigen, as measured by at least oneof a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to CD3, the disclosure providesvariant VH and VL domains. In one embodiment, the variant VH and VLdomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental VH and VL domain, as long as the ABD is stillable to bind to the target antigen, as measured at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantVH and VL are at least 90, 95, 97, 98 or 99% identical to the respectiveparental VH or VL, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

VI. Nucleic Acids

The disclosure further provides nucleic acid compositions encoding theanti-PSMA antibodies provided herein, including, but not limited to,anti-PSMA×anti-CD3 bispecific antibodies and anti-PSMA monospecificantibodies.

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format and scaffold of the heterodimericprotein. Thus, for example, when the format requires three amino acidsequences, such as for the 1+1 Fab-scFv-Fc format (e.g. a first aminoacid monomer comprising an Fc domain and a scFv, a second amino acidmonomer comprising a heavy chain and a light chain), three nucleic acidsequences can be incorporated into one or more expression vectors forexpression. Similarly, some formats (e.g. dual scFv formats such asdisclosed in FIG. 1) only two nucleic acids are needed; again, they canbe put into one or two expression vectors.

As is known in the art, the nucleic acids encoding the components of theantibodies described herein can be incorporated into expression vectorsas is known in the art, and depending on the host cells used to producethe heterodimeric antibodies described herein. Generally, the nucleicacids are operably linked to any number of regulatory elements(promoters, origin of replication, selectable markers, ribosomal bindingsites, inducers, etc.). The expression vectors can be extra-chromosomalor integrating vectors.

The nucleic acids and/or expression vectors of the antibodies describedherein are then transformed into any number of different types of hostcells as is well known in the art, including mammalian, bacterial,yeast, insect and/or fungal cells, with mammalian cells (e.g. CHOcells), finding use in many embodiments.

In some embodiments, nucleic acids encoding each monomer and theoptional nucleic acid encoding a light chain, as applicable depending onthe format, are each contained within a single expression vector,generally under different or the same promoter controls. In embodimentsof particular use in the antibodies described herein, each of these twoor three nucleic acids are contained on a different expression vector.As shown herein and in 62/025,931, hereby incorporated by reference,different vector ratios can be used to drive heterodimer formation. Thatis, surprisingly, while the proteins comprise first monomer:secondmonomer:light chains (in the case of many of the embodiments herein thathave three polypeptides comprising the heterodimeric antibody) in a1:1:2 ratio, these are not the ratios that give the best results.

The heterodimeric antibodies described herein are made by culturing hostcells comprising the expression vector(s) as is well known in the art.Once produced, traditional antibody purification steps are done,including an ion exchange chromatography step. As discussed herein,having the pIs of the two monomers differ by at least 0.5 can allowseparation by ion exchange chromatography or isoelectric focusing, orother methods sensitive to isoelectric point. That is, the inclusion ofpI substitutions that alter the isoelectric point (pI) of each monomerso that such that each monomer has a different pI and the heterodimeralso has a distinct pI, thus facilitating isoelectric purification ofthe “1+1 Fab-scFv-Fc” and “2+1” heterodimers (e.g., anionic exchangecolumns, cationic exchange columns). These substitutions also aid in thedetermination and monitoring of any contaminating dual scFv-Fc and mAbhomodimers post-purification (e.g., IEF gels, cIEF, and analytical IEXcolumns).

VII. Biological and Biochemical Functionality of the HeterodimericBispecific Antibodies

Generally the bispecific PSMA×CD3 antibodies described herein areadministered to patients with cancer, and efficacy is assessed, in anumber of ways as described herein. Thus, while standard assays ofefficacy can be run, such as cancer load, size of tumor, evaluation ofpresence or extent of metastasis, etc., immuno-oncology treatments canbe assessed on the basis of immune status evaluations as well. This canbe done in a number of ways, including both in vitro and in vivo assays.

VIII. Treatments

Once made, the compositions of the antibodies described herein find usein a number of applications. PSMA is highly expressed in prostatecancer. Accordingly, the heterodimeric compositions of the antibodiesdescribed herein find use in the treatment of such PSMA positivecancers.

IX. Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the antibodiesdescribed herein are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

X. Administrative Modalities

The antibodies and chemotherapeutic agents described herein areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time.

XI. Treatment Modalities

In the methods described herein, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MM) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the disclosure includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the disclosure aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies described herein depend on the disease or condition to betreated and may be determined by the persons skilled in the art.

An exemplary, non-limiting range for a therapeutically effective amountof an bispecific antibody used in the antibodies described herein isabout 0.1-100 mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the disclosure have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the antibodies describedherein. These examples are not meant to constrain the antibodiesdescribed herein to any particular application or theory of operation.For all constant region positions discussed in the antibodies describedherein, numbering is according to the EU index as in Kabat (Kabat etal., 1991, Sequences of Proteins of Immunological Interest, 5th Ed.,United States Public Health Service, National Institutes of Health,Bethesda, entirely incorporated by reference). Those skilled in the artof antibodies will appreciate that this convention consists ofnonsequential numbering in specific regions of an immunoglobulinsequence, enabling a normalized reference to conserved positions inimmunoglobulin families. Accordingly, the positions of any givenimmunoglobulin as defined by the EU index will not necessarilycorrespond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

Example 1: Generating Biologically Relevant Surrogates forPSMA-Expressing Tumor Cells

To ensure cell lines with biologically valid PSMA antigen densities wereused to inform the development of the novel anti-PSMA×anti-CD3bispecific antibodies of the invention, IHC was conducted on paraffinembedded arrays of 160 biopsy cores of prostate cancer, 16 tumoradjacent cores, 16 normal prostate cores, and several cancer cell lines.Illustrative IHC of biopsy cores are depicted in FIG. 12, and sampleswere qualitatively scored in-house on a scale of 0-3 with 0 representinglittle to no PSMA expression and 3 representing high PSMA expression(herein referred to as IHC score; breakdown of score for each core isdepicted in FIG. 13). Based on the results, it was determined that thenovel bispecific antibodies of the invention should target cell lineshaving IHC scores of 3, 2, and 1 as they represent 97% of the patientsegment and should not target score 0 cell lines as they resemble normaltissue. Upon matching the staining intensity between the sample types,cell lines were identified that could serve as surrogates of tumor andnormal tissues. LNCaP cancer cells were found to stain as intensely ascancer tumors expressing high amounts of PSMA; 22Rv1 cancer cells werefound to stain similar to some tumors expressing moderate amounts ofPSMA as well as some normal prostate cores; Huh-7 cancer cells werefound to express low levels of PSMA; and A549, ASPC-1, HT29, SKOV3, andPC3 cancer cells were found to express little to no PSMA.

PC3 cell lines expressing varying PSMA antigen densities were alsogenerated in order to expand the selection of surrogate cell lineshaving varying antigen density levels to tune the antibodies of theinvention. Cell-surface PSMA antigen density levels on the above cancercells lines and the PSMA-transfected PC3 cell lines were estimated byFACS using fluorescently-labeled beads as advised by the QuickCalprotocol (Bangs Laboratories, Inc., Fishers, Ind.). 50,000 cells perwell and MESF beads were combined with A647-conjugated anti-PSMA mAb(J591) for 30 minutes at 4° C. Cells were then washed and fixed in 1%PFA. Flow cytometry was performed to determine antibody binding, andantigen density calculations were carried out using QuickCal® V.2.3.software (Bangs Laboratories, Inc., Fishers, Ind.). Data depicting PSMAdensity on the various cell lines are depicted in FIGS. 14-16.PSMA-transfected PC3 cell lines are hereon referred to by their PSMAdensity (i.e. PC3 (˜100 k) has a MESF score of 100,000).

Example 2: Antigen Binding Domains

2A: CD3 Binding Domains

Sequences for CD3 binding domains having different CD3 bindingaffinities are depicted in FIG. 10.

2B: PSMA Binding Domains

The variable regions of a mouse anti-human PSMA binding domain werehumanized using string content optimization (see, e.g., U.S. Pat. No.7,657,380, issued Feb. 2, 2010). Sequences for the humanized PSMAbinding domain, hereon referred to as PSMA-H, are depicted in FIG. 17.

2B(a): Tuning PSMA-H Binding Affinity for PSMA

Variants of PSMA-H were engineered by introducing point substitutionsinto the variable light region (VL). In a first round, 75 variant VLswere engineered designated as L1.1-L1.75, sequences for which aredepicted in FIG. 18. The variant VLs were paired with the wild-typehumanized variable heavy region (VH) of PSMA-H and produced as bivalentmAbs (sequences for which are depicted in FIG. 19). Binding affinity ofthe variants for human PSMA was screened using Octet, a BioLayerInterferometry (BLI)-based method. Experimental steps for Octetgenerally include the following: Immobilization (capture of ligand to abiosensor); Association (dipping of ligand-coated biosensors into wellscontaining the analyte); and Dissociation (returning of biosensors towell containing buffer). In particular, anti-human Fc sensors were usedto capture the bivalent mAbs and dipped into human PSMA antigen. Theresulting apparent dissociation constant (KDapp due to avidityconcerns), association rate (ka), dissociation rate (kd), as well assensorgram response are depicted in FIG. 20.

Based on the above, useful variant VLs were identified on the basis ofchange in binding affinity for PSMA, reversion of CDRs to human germlinesequence (to reduce immunogenic potential), and introduction of negativecharge (to aid in longer serum half-life by reducing non-specificclearance). In some instances, substitutions from suitable variant VLswere combined to generate additional VL variants (sequences for whichare depicted in FIG. 18 as L1.76-L1.84). The variant VLs were pairedwith the wild-type humanized VH and produced as anti-PSMA×anti-CD3 bsAbs(sequences for which are depicted in FIG. 22) and screened for bindingaffinity for human and cynomolgus PSMA using Octet as generallydescribed above. The resulting dissociation constant (K_(D)),association rate (k_(a)), and dissociation rate (k_(d)) are depicted inFIGS. 34-35. A range of affinities from 6.55 nM to 116 nM were obtained.Notably, affinity for human and cynomolgus PSMA did not track across allthe variants. For example, XENP33756 bound human PSMA with K_(D) of 18nM while binding cynomolgus PSMA with K_(D) of 88 nM. For ease ofclinical development, it is advantageous for the PSMA binding domain tobind human and cynomolgus PSMA with similar affinity.

2C: Engineering Anti-PSMA×Anti-CD3 Bispecific Antibodies (bsAbs)

A number of formats for αPSMA×αCD3 bispecific antibodies (bsAbs) wereconceived, illustrative formats for which are outlined below and in FIG.21.

One such format is the 1+1 Fab-scFv-Fc format which comprises asingle-chain Fv (“scFv”) covalently attached to a first heterodimeric Fcdomain, a heavy chain variable region (VH) covalently attached to acomplementary second heterodimeric Fc domain, and a light chain (LC)transfected separately so that a Fab domain is formed with the variableheavy domain.

Another format is the 2+1 Fab2-scFv-Fc format which comprises a VHdomain covalently attached to a CH1 domain covalently attached to anscFv covalently attached to a first heterodimeric Fc domain(VH-CH1-scFv-Fc), a VH domain covalently attached to a complementarysecond heterodimeric Fc domain, and a LC transfected separately so thatFab domains are formed with the VH domains.

DNA encoding chains of the αPSMA×αCD3 bsAbs were generated by standardgene synthesis followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector containing fusion partners(e.g. domain linkers as depicted in FIG. 6 and/or backbones as depictedin FIGS. 7-9). DNA was transfected into HEK293E cells for expression.Sequences for illustrative αPSMA×αCD3 bsAbs (based on CD3 bindingdomains as described in Example 2A and PSMA binding domains as describedin Example 2B) in the 1+1 Fab-scFv-Fc format and in the 2+1 Fab2-scFv-Fcformat are depicted respectively in FIGS. 22-32.

Example 3: Prototypic 1+1 Anti-PSMA×Anti-CD3 bsAbs Indiscriminately KillCell Lines Expressing Low Levels of PSMA

To benchmark the activity of the novel anti-PSMA×anti-CD3 bsAbs of theinvention, the activity prototypic 1+1 anti-PSMA×anti-CD3 bsAbsXENP14484, XENP34282, and XENP34283 (sequences for which arerespectively depicted in FIGS. 22 and 33) were first investigated inredirected T-cell cytotoxicity (RTCC) assays.

PC3 cell lines expressing various PSMA densities (as described inExample 1) were transduced to constitutively express luciferase.Luciferase released from dead cancer cells rapidly degrade in assaymedia, so live target cells can be quantified based on luminescencereadout. The cancer cells were incubated with freshly enriched CD3⁺ Tcells at an effector to target ratio of 1:1 for 24 hours. Next, thebispecific antibodies were added to the cells at the indicatedconcentrations. In a first experiment, 48 hours after addition of thebsAbs, Bio-Glo Luciferase reagent (Promega, Madison, Wis.) was added andplates were read with the Envision Reader on luminescence setting. %RTCC was calculated by 1−(Raw Value/PBS AVG))*100. In a secondexperiment, 72 hours after addition of the bsAbs, cells were assayed viaflow cytometry for Ki67 expression on T cells (as an indicator ofproliferation).

Data showing cell kill are depicted in FIG. 36, and data showing T cellproliferation are depicted in FIG. 37. The data show that the twoprototypic 1+1 anti-PSMA×anti-CD3 induced RTCC on cell lines expressinghigh and low PSMA levels, including PC3 (˜3K) which represent normaltissues. In fact, in an in vivo study (data not shown), cynomolgusmonkeys treated with XENP14484 experienced dose-limiting toxicity (DLT;as indicated by high IL-6 release). Accordingly, activity on the PC3(˜3K) cell line may be a surrogate for DLT, and novel bispecificantibodies of the invention should be designed to avoid activity on PC3(˜3K) cells.

In another experiment, CD107a T cell degranulation was investigated asan indicator of activity by the prototypic bispecific antibodies. PC3cell lines expressing various PSMA densities (˜100K vs ˜50K) wereincubated with freshly enriched CD3⁺ T cells at an effector to targetratio of 10:1 for 24 hours. Next, the bispecific antibodies were addedto the cells at the indicated concentrations. 18 hours after addition ofthe bsAbs, cells were assayed via flow cytometry for CD107adegranulation on T cells, data for which are shown in FIG. 46. The datashow that each of the prototypic bispecific antibodies induced T celldegranulation with very similar potency in the presence of both cellshaving higher and lower PSMA densities.

Example 4: Tuning Anti-PSMA×Anti-CD3 bsAbs to Enhance Selectivity andTherapeutic Index

4A: Tuning PSMA Binding Valency and Binding Affinity

In order to encourage avid binding and strong activity on high PSMAexpressing cells (e.g. tumors) while minimizing reactivity on lowexpressing cells (e.g. normal tissues), the anti-PSMA×anti-CD3 bsAbswere tuned for PSMA binding valency and affinity as well as CD3 bindingaffinity. Towards this, anti-PSMA×anti-CD3 bsAbs were engineered in the2+1 Fab₂-scFv-Fc format with a range of PSMA binding affinities andreduced CD3 binding affinities and the following illustrative bsAbs wereinvestigated in cell binding and RTCC assays: XENP31855 (having 1 nMK_(D) PSMA-H H1L1 and CD3 High-Int#1[VLVH]), XENP32218 (having 7 nMPSMA-H_H1_L1.58 and CD3 High-Int#1[VLVH]), XENP32220 (having 38 nMPSMA-H_H1_L1.24 and CD3 High-Int#1[VLVH]), and XENP32224 (having 83 nMPSMA-H H1_L1.29 and CD3 High-Int#1[VLVH]).

First, the effect of reduced monovalent PSMA binding affinity on cellbinding was investigated. PSMA-transfected PC3 (˜32K) cancer cells weretreated with the indicated concentrations of the indicated testarticles. Binding was detected using anti-human Fc mAb, data for whichare shown in FIG. 38. The data show that in comparison the monovalent1+1 Fab-scFv-Fc format, the bivalent 2+1 Fab2-scFv-Fc format retainscell binding despite reduced monovalent PSMA binding affinity due toavidity.

Next, cell lines expressing various PSMA densities were transduced toconstitutively express luciferase. The cells were incubated with freshlyenriched CD3⁺ T cells at an effector to target ratio of 1:1 for 24hours. Next, the bispecific antibodies were added to the cells at theindicated concentrations. 18 hours after addition of the bsAbs, cellswere assayed via flow cytometry for CD107a degranulation on T cells (asan indicator of T cell activity). 48 hours after addition of the bsAbs,Bio-Glo Luciferase reagent (Promega, Madison, Wis.) was added and plateswere read with the Envision Reader on luminescence setting. % RTCC wascalculated by 1−(Raw Value/PBS AVG))*100. 72 hours after addition of thebsAbs, cells were assayed via flow cytometry for Ki67 expression on Tcells (as an indicator of proliferation). Data showing cell kill aredepicted in FIG. 39, data showing T cell proliferation are depicted inFIG. 40, and data showing CD107a degranulation are depicted in FIG. 41.Collectively, the data show that as PSMA binding affinity is reduced,selectivity for higher antigen density cell lines over lower antigendensity cell lines is improved. Notably as shown in FIGS. 42 and 39,with low PSMA binding affinity (e.g. 38 nM and 83 nM) it is possible toachieve efficacious killing of cell lines exhibiting IHC Score 2 andScore 1 while inducing little to no killing of cell line exhibiting IHCScore 0.

4B: Further Tuning CD3 Binding Affinity

Another approach explored for optimizing the anti-PSMA×anti-CD3bispecific antibodies was tuning CD3 binding affinity. According,anti-PSMA×anti-CD3 bsAbs in the 2+1 Fab₂-scFv-Fc format having CD3High-Int#2[VLVH] binding domain and either 1 nM KD or 7 nM KD PSMAbinding domains, respectively XENP31856 and XENP33063, were investigatedin assays as generally described above. Data as depicted in FIGS. 43 and44 show that reducing the CD3 binding affinity may also convey selectivetargeting to high PSMA expressing cell lines (even with higher affinityPSMA binding).

4C: Tuned Anti-PSMA×Anti-CD3 Bispecific Antibodies are Able to KillClinically Relevant PSMA+Prostate Cancer Cells

To confirm that the tuned anti-PSMA×anti-CD3 bispecific antibodies ofthe invention are able to kill clinically relevant PSMA⁺ prostate cancercells, their ability to induce RTCC on LNCaP (human prostateadenocarcinoma cells having IHC score 3 and ˜140K PSMA density) and22Rv1 (human prostate carcinoma cells having IHC score 2 and ˜115K PSMAdensity).

Cancer cells were incubated with freshly enriched CD3⁺ T cells at aneffector to target ratio of 10:1 for 24 hours. Next, the bispecificantibodies were added to the cells at the indicated concentrations. 72hours after addition of the bsAbs, Bio-Glo Luciferase reagent (Promega,Madison, Wis.) was added and plates were read with the Envision Readeron luminescence setting. % RTCC was calculated by 1−(Raw Value/PBSAVG))*100. Data depicted in FIG. 45 show that tuned anti-PSMA×anti-CD3bispecific mAb XENP32220 was able to induce cell kill on both LNCaP and22Rv1 cancer cells.

Example 5: Tuned PSMA×CD3 Bispecific Antibodies are Active In Vivo

Next, the in vivo anti-tumor effect of the tune anti-PSMA×anti-CD3bispecific antibodies of the invention was investigated. NOD-SCID gamma(NSG) mice were engrafted intradermally with 1×10{circumflex over ( )}6PC3 (˜100K) cells in the right flank on Day −16. On Day −1, mice wereengrafted intraperitoneally with 5×10{circumflex over ( )}6 human PBMCs.Mice (n=10) were then treated on Days 0, 8, 15, and 22 with 3 mg/kgXENP32218, XENP32220, or XENP32224. Controls (N=10) used were PBS and 3mg/kg anti-PD-1 mAb (a checkpoint inhibitor which enhances anti-tumoractivity by de-repressing the engrafted human T cells). Tumor volumeswere monitored by caliper measurements, data for which are shown in FIG.47 for Day 19 and change over time in FIG. 48. The data show that eachof the tuned PSMA×CD3 bispecific antibodies significantly enhanced(p<0.05 vs. PBS or αPD-1 mAb) anti-tumor activity (as indicated by tumorvolume; statistics performed on baseline corrected data using unpairedt-test) despite their reduced PSMA binding affinity.

Example 6: Tuned PSMA×CD3 bsAbs Demonstrate Favorable Tolerability andPharmacokinetics in Cynomolgus

In a cynomolgus study, each healthy male cynomolgus (n=1) wasadministered by IV either a 1× dose, 10× dose, or 60× dose of tunedPSMA×CD3 bsAbs XENP32218, XENP32220, or XENP32224, or the analogs ofthese three molecules that additionally contain the Xtend mutation(M428L/N434S for enhanced serum half-life) in the Fc domain-XENP34626,XENP34627, or XENP34628, respectively. All XENPs were generally welltolerated (i.e., no dose limiting side effects) up to the highest 60×dose (data not shown). As seen in FIG. 49, the variants with the Xtendmutations resulted in improved pharmacokinetics, particularly at the 1×and 10× doses. For example, the terminal serum half-lives of XENP32220were 1.59, 3.01, or 7.95 days at each relative dose level, while itsXtend analog XENP34267 had serum half-lives of 4.5, 8.6, and 10.8 daysacross each dose level. Additionally, the lower PSMA affinity XENP32224had half-lives of 2.53 or 3.34 days at the 1× and 10× doses, while itsXtend analog XENP34628 had half-lives of 5.9 or 9.3 days at the 1× and10× dose levels. These half life measurements of the Xtend analog were asignificant improvement over half life data for a comparator PSMA×CD3bsAb molecule at a comparable dose (as reported in literature).

At the lowest 1× dose level, serum clearance was PSMAaffinity-dependent. XENP32218, having the highest PSMA binding affinity,demonstrated the fastest clearance, and XENP32224, having the lowestPSMA binding affinity, demonstrated the slowest clearance. Notably, the60× dose was high enough to clear the sink effect so that half lives atthat dose were no longer affinity-dependent.

1. A heterodimeric antibody comprising: a) a first monomer comprising,from N-terminus to C-terminus, a VH1-CH1-linker 1-scFv-linker 2-CH2-CH3,wherein VH1 is a first variable heavy domain, scFv is an anti-CD3 scFV,linker 1 and linker 2 are a first domain linker and second domainlinker, respectively, and CH2-CH3 is a first Fc domain; b) a secondmonomer comprising, from N-terminus to C-terminus, aVH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain andCH2-CH3 is a second Fc domain; and c) a common light chain comprising avariable light domain and a light chain constant domain; wherein thefirst variable heavy domain and the variable light domain form a firstPSMA binding domain, and the second variable heavy domain and thevariable light domain form a second PSMA binding domain.
 2. Aheterodimeric antibody according to claim 1, wherein the first andsecond PSMA binding domains each comprise the variable heavycomplentarity determining regions vhCDR1, vhCDR2 and vhCDR3 of PSMA-Hvariable heavy domain H1 (SEQ ID NO: 217); and the variable lightcomplementarity determining regions (vlCDR1, vlCDR2 and clCDR3) of aPSMA-H variable light domain selected from any one of the PSMA-Hvariable light domains L1.24 (SEQ ID NO: 252), L1.29 (SEQ ID NO: 257),and L1.58 (SEQ ID NO: 286).
 3. A heterodimeric antibody according toclaim 1, wherein the first and second variable heavy domain each is aPSMA-H variable heavy domain H1 (SEQ ID NO: 217), and the variable lightdomain is selected from any one of the PSMA-H variable light domainsL1.24 (SEQ ID NO: 252), L1.29 (SEQ ID NO: 257), and L1.58 (SEQ ID NO:286).
 4. A heterodimeric antibody according to claim 1, wherein the scFvcomprises the variable heavy complementarity determining regions vhCDR1(SEQ ID NO: 167), vhCDR2 (SEQ ID NO: 168), vhCDR3 (SEQ ID NO: 169) andthe variable light complementarity determining regions vlCDR1 (SEQ IDNO: 163), vlCDR2 (SEQ ID NO: 164), and vlCDR3 (SEQ ID NO: 165) ofL1.47_H1.32 (SEQ ID NO 161).
 5. A heterodimeric antibody according toclaim 1, wherein the scFv comprises the variable heavy domain andvariable light domain of L1.47_H1.32 (SEQ ID NO 161). 6.-22. (canceled)23. A heterodimeric antibody according to claim 1, selected from thegroup consisting of: a) XENP34626, comprising a first chain having thesequence of SEQ ID NO: 556, a second chain having the sequence of SEQ IDNO: 557, and a third chain having a sequence of SEQ ID NO: 558; b)XENP34627, comprising a first chain having the sequence of SEQ ID NO:559, a second chain having the sequence of SEQ ID NO: 560, and a thirdchain having a sequence of SEQ ID NO: 561; and c) XENP34628, comprisinga first chain having the sequence of SEQ ID NO: 562, a second chainhaving the sequence of SEQ ID NO: 563, and a third chain having asequence of SEQ ID NO:
 564. 24. A nucleic acid composition comprising:a) a first nucleic acid encoding the first monomer according to claim 1;b) a second nucleic acid encoding the second monomer according to claim1; c) a third nucleic acid encoding the common light chain according toclaim
 1. 25. An expression vector composition comprising: a) a firstexpression vector comprising the first nucleic acid according to claim24; b) a second expression vector comprising the second nucleic acidaccording to claim 24; and c) a third expression vector comprising thethird nucleic acid according to claim
 24. 26. A host cell transformedwith the expression vector composition according to claim
 25. 27.-53.(canceled)
 54. A composition comprising a Prostate Specific MembraneAntigen (PSMA) binding domain comprising: a) a variable heavy domaincomprising the variable heavy complementarity determining regionsvhCDR1, vhCDR2, and vhCDR3 of PSMA-H variable heavy domain H1 (SEQ IDNO: 217); and b) a variable light domain comprising the variable lightcomplementarity determining regions vlCDR1, vlCDR2, and vlCDR3 of aPSMA-H variable light domain selected from PSMA-H variable light domainsL1.24 (SEQ ID NO: 252), L1.29 (SEQ ID NO: 257), and L1.58 (SEQ ID NO:286).
 55. A composition comprising a Prostate Specific Membrane Antigen(PSMA) binding domain comprising: a) a variable heavy domain, whereinthe variable heavy domain is the PSMA-H variable heavy domain H1 (SEQ IDNO: 217); and b) a variable light domain selected from PSMA-H variablelight domains L1.24 (SEQ ID NO: 252), L1.29 (SEQ ID NO: 257), and L1.58(SEQ ID NO: 286). 56.-82. (canceled)